Muscle-derived cells (MDCs) for treating muscle- or bone-related injury or dysfunction

ABSTRACT

The present invention provides muscle-derived cells, preferably myoblasts and muscle-derived stem cells, genetically engineered to contain and express one or more heterologous genes or functional segments of such genes, for delivery of the encoded gene products at or near sites of musculoskeletal, bone, ligament, meniscus, cartilage or genitourinary disease, injury, defect, or dysfunction. Ex vivo myoblast mediated gene delivery of human inducible nitric oxide synthase, and the resulting production of nitric oxide at and around the site of injury, are particularly provided by the invention as a treatment for lower genitourinary tract dysfunctions. Ex vivo gene transfer for the musculoskeletal system includes genes encoding acidic fibroblast growth factor, basic fibroblast growth factor, epidermal growth factor, insulin-like growth factor, platelet derived growth factor, transforming growth factor-β, transforming growth factor-α, nerve growth factor and interleukin-1 receptor antagonist protein (IRAP), bone morphogenetic protein (BMPs), cartilage derived morphogenetic protein (CDMPs), vascular endothelial growth factor (VEGF), and sonic hedgehog proteins.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.09/302,896 filed on Apr. 30, 1999, which claims the benefit ofProvisional Patent Application U.S. Ser. No. 60/083,917, filed May 1,1998, the entire disclosures of which are hereby incorporated byreference.

GOVERNMENT INTERESTS

This invention was made with Government support under Grant No. DK055387awarded by the National Institutes of Health. The Government has certainrights to this invention.

FIELD OF THE INVENTION

The present invention relates generally to compositions and methodscomprising myogenic or muscle-derived cells, including myoblasts andmuscle-derived stem cells (also termed MDC herein) for tissueengineering and cell-mediated gene therapy. The invention furtherrelates to the introduction of exogenous nucleic acids intomuscle-derived cells, including myoblasts and muscle-derived stem cells,resulting in the expression of one or more gene products by thegenetically engineered muscle-derived cells. Such engineered cells arethen capable of producing the gene products and effecting an enhancedphysiological response after administration to a recipient host,including humans.

BACKGROUND OF THE INVENTION

A number of defects, diseases and pathological conditions in a varietyof areas of medicine would benefit from the development of noninvasivetreatments utilizing improved gene delivery vehicles and systems thatallow the safe, efficient and sustained production of gene products toan affected tissue or organ site. In particular, improved cell-mediatedgene delivery vehicles and methods would find wide use in amelioratingnon-fatal, yet debilitating, pathologies of the musculoskeletal system,such as arthritis and joint disease (e.g., ligament, meniscus andcartilage); the bone, such as segmental bone defects and non-unions; andthe genitourinary system, such as urinary incontinence and bladderconditions.

Although synovial cells have been used to deliver potentiallytherapeutic agents into the joint, the expression of such agents hasdeclined over time, thereby causing these agents generally to becomeundetectable after about four to six weeks. (G. Bandara et al., 1993,Proc. Natl. Acad. Sci. USA, 90(22):10764-10768; C. H. Evans and P. D.Robbins, 1995, Ann. Med., 27(5):543-546; C. H. Evans and P. D. Robbins,1994, J. Rheum., 21(5):779-782). This decline in expression over timemay be ameliorated by the use of cell mediated gene delivery employing amyogenic cell type that becomes post-mitotic with differentiation, inaccordance with the present invention.

Segmental bone defects and non-unions are relatively common problemsfacing all orthopedic surgeons. Osteogenic proteins, e.g., bonemorphogenic protein-2, BMP-2), can promote bone healing in segmentalbone defects. However, a large quantity of the human recombinant proteinis needed to enhance bone healing potential. Moreover, current modes ofdelivering such quantities of protein, i.e., a biological allograft or asynthetic carrier, are hampered by limited availability, possibledisease transmission and the need for further research andinvestigation.

Cell mediated gene therapy in the bone defect would allow a sustainedexpression of osteogenic proteins, further enhance bone healing, andoffer a solution to the problems surrounding current methods of boneprotein delivery. Thus, in accordance with the present invention, theutilization of muscle-derived cells, e.g., myoblasts, as cellular genedelivery vehicles to correct or improve a bone defect, provides animportant step in establishing a less invasive treatment for non-unionsand segmental bone defects.

Ex vivo gene therapy and myoblast transplantation are two closelyrelated methods which require in vitro cell isolation and culture. Exvivo techniques involve muscle biopsy and myogenic cell isolation (T. A.Rando et al., 1994, J. Cell Biol., 125:1275-1287; Z. Qu et al., 1998, J.Cell Biol., 142(5):1257-1267). The isolated muscle-derived cells aretransduced in vitro with the desired gene carrying vector. The satellitecells are then reinjected into skeletal muscle, fuse to formpost-mitotic myotubes and myofibers, and begin growth factor production.This technique is feasible with adenoviral, retroviral, and herpessimplex viral vectors.

The following are examples of orthopaedic applications for muscle basedgene therapy and tissue engineering related to the practice of thepresent invention:

Muscle Injury and Repair

Muscle injuries comprise a large percentage of recreational andcompetitive athletic injuries. Muscle injuries may result from bothdirect (e.g., contusions, lacerations) and indirect (e.g., strains,ischemia and neurological injuries) trauma. Upon injury, satellite cellsare released and activated in order to differentiate into myotubes andmyofibers, thereby promoting muscle healing. However, this reparativeprocess is usually incomplete and accompanied by a fibrous reactionproducing scar tissue. This scar tissue limits the muscle's potentialfor functional recovery (T. Hurme et al., 1991; Med. Sci. Sports Exerc.,23:801-810; T. Hurme et al., 1992, Med. Sci. Sports Exerc., 24:197-205).

Investigations in animals have identified possible clinical applicationsfor muscle-based tissue engineering to treat muscle injuries (W. E.Garrett et al., 1984; J. Hand Surgery (Am). 9A:683-692; W. E. Garrett etal., 1990, Med. Sci. Sports Exerc., 22:436-443). Injured skeletal musclereleases numerous growth factors acting in autocrine and paracrinefashion to modulate muscle healing. These proteins activate satellitecells to proliferate and differentiate into myofibers (T. Hurme, 1992,Med. Sci. Sports Exerc., 24:197-205; R. Bischoff, 1994, “The satellitecell and muscle regeneration”. Myology. 2nd Edition. New York,McGraw-Hill, Inc, pp. 97-118; H. S. Allamedine et al., 1989; MuscleNerve, 12:544-555; E. Schultz et al., 1985, Muscle Nerve, 8:217; E.Schultz, 1989, Med. Sci. Sports Exerc., 21:181).

Muscle-based tissue engineering offers exciting potential therapies formuscle disorders. A large number of recreational and professionalathletic injuries involve skeletal muscle (Garrett et al., 1990, Med.Sci. Sports Exerc., 22:436-443). Therapies to improve functionalrecovery and shorten rehabilitation may both optimize performance andminimize morbidity. Further research is ongoing to refine thesemuscle-based tissue engineering applications. The results of suchinvestigations may provide revolutionary treatments for these commonmuscle injuries. The present invention provides new and excitingtreatments for muscle repair following muscle-based injuries,particularly for application in clinical settings.

Bone Healing

Multiple surgical specialties, including orthopaedic, plastic, andmaxillofacial, are concerned with bone healing augmentation. Physiciansin these disciplines rely on bone augmentation techniques to improvehealing of fracture non-unions, oncologic and traumatic bone defectreconstructions, joint and spine fusions, and artificial implantstabilizations. Unfortunately, current techniques of autograft,allograft, and electrical stimulation are often suboptimal. Therefore,tissue engineering approaches toward bone formation have immenseimplications.

Intramuscular bone formation is a poorly understood phenomenon. It canbe present in the clinically pathologic states of heterotopicossification, myositis ossificans, fibrodysplasia ossificans progressivaand osteosarcoma. Radiation therapy and the anti-inflammatory drug,indomethicin, can suppress myositis ossificans. However, neither themechanism of formation nor suppression of ectopic bone is clearlyunderstood. A growing family of bone morphogenetic proteins (BMPs),members of the transforming growth factor β (TGF-β) superfamily, arerecognized as being capable of stimulating intramuscular bone. HumanBMP-2 in recombinant form (rhBMP-2) and BMP-encoding cDNA contained in aplasmid construct induce bone formation when injected into skeletalmuscle (E. A. Wang et al., 1990, Proc. Natl. Acad. Sci. USA,87:2220-2224; J. Fang et al, 1996, Proc. Natl. Acad. Sci. USA,93:5753-5758). Current applications focus on injecting rhBMP-2 directlyinto non-unions and bone defects. However, muscle-based tissueengineering has enormous promise in the arena of bone healing and mayshed light on the physiologic mechanism of ectopic bone formation.

Intraarticular Disorders

Degenerative and traumatic joint disorders are encountered frequently asour population becomes more active and lives longer. These disordersinclude arthritis of various etiologies, ligament disruptions, meniscaltears, and osteochondral injuries. Currently, the clinician's toolsconsist primarily of surgical procedures aimed at biomechanicallyaltering the joint, such as anterior cruciate ligament (ACL)reconstructions, total knee replacement, meniscal repair or excision,cartilage debridement, etc. Tissue engineering applied to theseintraarticular disease states theoretically offers a more biologic andless disruptive reparative process.

Both direct (I. Nita et al., 1996, Arthritis Rheum., 39:820-828) and exvivo (G. Bandara et al., 1993, Proc. Natl. Acad. Sci. USA,90:10764-10768) gene therapy approaches to arthritis models have beenreported. The synovial cell-mediated ex vivo approach, while offeringadvantages of ex vivo gene transfer such as the safety of in vitrogenetic manipulation and precise cell selection, is hindered by adecline of gene expression after 5-6 weeks (Bandara et al., 1993,Ibid.). Due to its ability to form post-mitotic myotubes and myofibers,the satellite cell offers the theoretical advantages of longer term andmore abundant protein production.

Muscle cell-mediated ex vivo gene delivery to numerous intraarticularstructures is possible. Intraarticular injection of primary myoblasts,transduced by adenovirus carrying the β-galactosidase marker gene,results in gene delivery to many intraarticular structures (C. S. Day etal., 1997, J. Orthop. Res., 15:227-234). Tissues expressingβ-galactosidase at 5 days after injection in the rabbit knee include thesynovial lining, meniscal surface, and cruciate ligament (Ibid.). Incontrast, injection of transduced synovial cells results inβ-galactosidase expression only in the synovium (Ibid.). Likewise,injection of transduced immortalized myoblasts results in gene deliveryto various intraarticular structures, including the synovial lining andpatellar ligament surface. However, the purified immortalized myoblastsfused more readily and resulted in more de novo intraarticular myofibersthan the primary myoblasts. This illustrates the importance of obtaininga pure population of myogenic cells, void of the fibroblast andadipocyte contamination often seen in primary myoblasts.

Muscle cell-mediated ex vivo approaches are predicated on myoblastfusion to form myofibers, the plurinuclear protein-producing factories.Intraarticular injection of transduced immortalized myoblasts into asevere combined immune deficient (SCID) mouse results in myotubeformation and transgene expression in multiple structures at 35 days.Therefore, intraarticular gene expression (for at least 35 days)resulting from muscle cell-mediated tissue engineering is feasible inanimal models. Based on this data, a muscle cell-mediated gene transferapproach may deliver genes to improve the healing of severalintraarticular structures specifically to the ACL and meniscus.

The ACL is the second most frequently injured knee ligament.Unfortunately, the ACL has a low healing capacity, possibly secondary toits encompassing synovial sheath or the surrounding synovial fluid.Because complete tears of the ACL are incapable of spontaneous healing,current treatment options are limited to surgical reconstruction usingautograft or allograft. The replacement graft, often either patellaligament or hamstring tendon in origin, undergoes ligamentization witheventual collagen remodeling (S. P. Arnosczky et al., 1982, Am. J.Sports Med., 10:90-95). Therefore, augmentation of this ligamentizationprocess using growth factors to affect fibroblast behavior is envisionedby the practice of the methods described herein. In vivo data suggeststhat platelet-derived growth factor (PDGF), transforming growth factor-β(TGF-β), and epidermal growth factor (EGF) promote ligament healing (N.A. Conti et al., 1993, Trans. Orthop. Res. Soc., 18:60). Transient, lowlevels of these growth factors resulting from their direct injectioninto the injured ligament are unlikely to produce a significantresponse. Therefore, an efficient delivery mechanism is essential to thedevelopment of a clinically applicable therapy. Muscle cell-mediated exvivo gene therapy according to the teachings herein offers the potentialto achieve persistent local gene expression and subsequent growth factordelivery to the ACL.

With more specific regard to the knee, the knee meniscus plays acritical role in maintaining normal knee biomechanics. Primary functionsof the meniscus include load transmission, shock absorption, jointlubrication, and tibiofemoral stabilization in the ACL deficient knee.The historical treatment of menisectomy for meniscal tears has beenreplaced by meniscal repair when tears involve the meniscus' peripheral,vascular third. Growth factors, including platelet-derived growth factor(PDGF), are capable of enhancing meniscal healing (K. P. Spindler etal., 1995, J. Orthop. Res., 13(2):201-207). However, needed for both thepractitioner and the patient are better methods and procedures todeliver such needed factors to the meniscus to provide healing andrepair.

Urologic Applications

Urinary incontinence is a devastating medical and social condition. Theincidence of urinary incontinence is increasing in the United States dueto an aging population. As of January 1997, the National Institute ofDiabetes and Digestive and Kidney Disease has launched a public healthcampaign to address the fact that there are over eleven million womenand four million men in the United States who have urinary incontinenceproblems. Approximately half of the fifteen million people withincontinence have stress urinary incontinence; however, less than halfof the afflicted people are seeking help and receiving the treatmentswhich are available (Agency for Health Care Policy and Research, AHCPR,1992 and 1996).

Presently, the estimated annual cost for treating people with urinaryincontinence is over $16 billion in the United States. Most of thismoney is spent on management measures, such as adult diapers and pads,rather than on treatment. Since most of the invasive and surgicaltreatment for urinary incontinence involves the treatment of stressurinary incontinence, the cost for managing stress urinary incontinenceis estimated at $9 billion dollars per year in the United States (AHCPR1996).

In evaluating an individual with incontinence, three of the most commontypes and causes of incontinence can be identified: a) urgeincontinence, b) stress incontinence, or c) overflow incontinence (M. B.Chancellor and J. G. Blaivas, 1996, Atlas of Urodynamics, Williams andWilkins, Philadelphia, Pa.).

Stress incontinence is the involuntary loss of urine during coughing,sneezing, laughing, or other physical activities which increaseabdominal pressure. This condition may be confirmed by observing urineloss coincident with an increase in abdominal pressure, in the absenceof a bladder contraction or an overdistended bladder. The condition ofstress incontinence may be classified as either urethral hypermobilityor intrinsic sphincter deficiency. In urethral hypermobility, thebladder neck and urethra descend during cough or strain on urodynamicand the urethra opens with visible urinary leakage (leak point pressurebetween 60-120 cm H₂O). In intrinsic sphincter deficiency, the bladderneck opens during bladder filling without bladder contraction. Visibleurinary leakage is seen with minimal or no stress. There is variablebladder neck and urethral descent, often none at all, and the leak pointpressure is low (<60 cm H₂O). (J. G. Blaivas, 1985, Urol. Clin. N.Amer., 12:215-224; D. R. Staskin et al., 1985, Urol. Clin. N. Amer.,12:271-278).

Urge incontinence is defined as the involuntary loss of urine associatedwith an abrupt and strong desire to void. Although involuntary bladdercontractions can be associated with neurologic disorders, they can alsooccur in individuals who appear to be neurologically normal (P. Abramset al., 1987, Neurol. & Urodynam., 7:403-427). Common neurologicdisorders associated with urge incontinence are stroke, diabetes, andmultiple sclerosis (E. J. McGuire et al, 1981, J. Urol., 126:205-209).Urge incontinence is caused by involuntary detrusor contractions thatcan also be due to bladder inflammation and impaired detrusorcontractility where the bladder does not empty completely.

Overflow incontinence is characterized by the loss of urine associatedwith overdistension of the bladder. Overflow incontinence may be due toimpaired bladder contractility or to bladder outlet obstruction leadingto overdistension and overflow. The bladder may be underactivesecondarily to neurologic conditions such as diabetes or spinal cordinjury, or following radical pelvic surgery.

Another common and serious cause of urinary incontinence (urge andoverflow type) is impaired bladder contractility. This is anincreasingly common condition in the geriatric population and inpatients with neurological diseases, especially diabetes mellitus (N. M.Resnick et al., 1989, New Engl. J. Med., 320:1-7; M. B. Chancellor andJ. G. Blaivas, 1996, Atlas of Urodynamics, Williams and Wilkins,Philadelphia, Pa.). With inadequate contractility, the bladder cannotempty its content of urine; this causes not only incontinence, but alsourinary tract infection and renal insufficiency. Presently, cliniciansare very limited in their ability to treat impaired detrusorcontractility. There are no effective medications to improve detrusorcontractility. Although urecholine can slightly increase intravesicalpressure, it has not been shown in controlled studies to aid effectivebladder emptying (A. Wein et al., 1980, J. Urol., 123:302). The mostcommon treatment is to circumvent the problem with intermittent orindwelling catheterization.

There are a number of treatment modalities for stress urinaryincontinence. The most commonly practiced current treatments for stressincontinence include the following: absorbent products; indwellingcatheterization; pessary, i.e., vaginal ring placed to support thebladder neck; and medication (Agency for Health Care Policy andResearch. Public Health Service: Urinary Incontinence Guideline Panel.Urinary Incontinence in Adults: Clinical Practice Guideline. AHCPR Pub.No. 92-0038. Rockville, Md. U.S. Department of Health and HumanServices, March 1992; M. B. Chancellor, Evaluation and Outcome. In: TheHealth of Women With Physical Disabilities: Setting a Research Agendafor the 90's. Eds. Krotoski D. M., Nosek, M., Turk, M., BrooksPublishing Company, Baltimore, Md., Chapter 24, 309-332, 1996). Withspecific regard to medication, there are several drugs approved for thetreatment of urge incontinence. However, there are no drugs approved oreffective for stress urinary incontinence.

Exercise is another treatment modality for stress urinary incontinence.For example, Kegel exercise is a common and popular method to treatstress incontinence. The exercise can help half of the people who can doit four times daily for 3-6 months. Although 50% of patients report someimprovement with Kegel exercise, the cure rate for incontinencefollowing Kegel exercise is only 5 percent. In addition, most patientsstop the exercise and drop out from the protocol because of the verylong time and daily discipline required.

Another treatment method for urinary incontinence is the urethral plug.This is a new, inexpensive disposable cork-like plug for women withstress incontinence. A new plug should be used after each micturition,with an estimated daily cost of about $15-20. The estimated annualdisposable cost is over $5,000. The plug is associated with over 20%urinary tract infection and, unfortunately, does not cure incontinence.

Biofeedback and functional electrical stimulation using a vaginal probeare also used to treat urge and stress urinary incontinence. However,these methods are time-consuming and expensive and the results are onlymoderately better than Kegel exercise. Surgeries, such as laparoscopicor open abdominal bladder neck suspensions; transvaginal approachabdominal bladder neck suspensions; artificial urinary sphincter(expensive complex surgical procedure with 40% reversion rate) are alsoused to treat stress urinary incontinence.

Other treatments include urethra injection procedures with exogenousinjectable materials such as Teflon, collagen, and autologous fat. Eachof these injectables has its disadvantages. More specifically, there aresignificant reservations among those in the medical community concerningthe use of Teflon. Complications of Teflon injection include granuloma,diverticulum, cysts, and urethral polyp formation. Of greatest concernis the migration (via the lymphatic and vascular systems) of Teflonparticles to distant locations, resulting in fever and pneumonitis.

Collagen injections generally employ bovine collagen, which is expensiveand is often reabsorbed, resulting in the need for repeated injections.A further disadvantage of collagen is that about 5% of patients areallergic to bovine source collagen and develop antibodies.

Autologous fat grafting as an injectable bulking agent has a significantdrawback in that most of the injected fat is resorbed. In addition, theextent and duration of the survival of an autologous fat graft remainscontroversial. An inflammatory reaction generally occurs at the site ofimplant. Complications from fat grafting include fat resorption, nodulesand tissue asymmetry.

In view of the above-mentioned limitations and complications of treatingurinary incontinence and bladder contractility, new and effectivemodalities in this area are needed in the art. In accordance with thepresent invention, muscle cell injection therapy using uniquelyengineered muscle-derived cells is provided as an improved and novelmeans for treating and curing various types of incontinence,particularly, stress urinary incontinence and for the enhancement ofurinary continence. As but one advantage, muscle-derived cell injectioncan preferably be autologous, so that there will minimal or no allergicreactions, unlike the aforementioned use of collagen. Also, unlikecollagen, myogenic cells such as blasts are not absorbed; thus, they canprovide a better improvement and cure rate.

Myoblasts, the precursors of muscle fibers, are mononucleated musclecells which differ in many ways from other types of cells. Myoblastsnaturally fuse to form post-mitotic multinucleated myotubes which resultin the long-term expression and delivery of bioactive proteins (T. A.Partridge and K. E. Davies, 1995, Brit. Med. Bulletin, 51:123-137; J.Dhawan et al., 1992, Science, 254: 1509-1512; A. D. Grinnell, 1994, In:Myology. Ed 2, Ed. Engel AG and Armstrong CF, McGraw-Hill, Inc, 303-304;S. Jiao and J. A. Wolff, 1992, Brain Research, 575:143-147; H.Vandenburgh, 1996, Human Gene Therapy, 7:2195-2200). Myoblasts have beenused for gene delivery to muscle for muscle-related diseases, such asDuchenne muscular dystrophy (E. Gussoni et al., 1992, Nature,356:435-438; J. Huard et al., 1992, Muscle & Nerve, 15:550-560; G.Karpati et al., 1993, Ann. Neurol., 34:8-17; J. P. Tremblay et al.,1993, Cell Transplantation, 2:99-112), as well as for non-muscle-relateddiseases, e.g., gene delivery of human adenosine deaminase for theadenosine deaminase deficiency syndrome (C. M. Lynch et al., 1992, Proc.Natl. Acad. Sci. USA, 89:1138-1142); gene transfer of human proinsulinfor diabetes mellitus (G. D. Simonson et al., 1996, Human Gene Therapy,7:71-78); gene transfer for expression of tyrosine hydroxylase forParkinson's disease (S. Jiao et al., 1993, Nature, 362:450); transferand expression of Factor IX for hemophilia B (Y. Dai et al., 1995, Proc.Natl. Acad. Sci. USA, 89:10892), delivery of human growth hormone forgrowth retardation (J. Dhawan et al., 1992, Science, 254:1509-1512).

The use of myoblasts to treat muscle degeneration, to repair tissuedamage or treat disease is disclosed in U.S. Pat. Nos. 5,130,141 and5,538,722. Also, myoblast transplantation has been employed for therepair of myocardial dysfunction (S. W. Robinson et al., 1995, CellTransplantation, 5:77-91; C. E. Murry et al., 1996, J. Clin. Invest.,98:2512-2523; S. Gojo et al., 1996, Cell Transplantation, 5:581-584; A.Zibaitis et al., 1994, Transplantation Proceedings, 26:3294).

Nitric oxide (NO) has been recognized as a important transmitter ingenitourinary tract function. NO mediates smooth muscle relaxation andis also the key to achieving erection. Recently, constitutive andinducible nitric oxide synthase (NOS or iNOS) have been demonstrated inthe urothelium, bladder and urethra wall. A deficiency in urinary NO inpatients having interstitial cystitis bladder inflammation (M. A.Wheeler et al., 1997, J. Urol., 158(6):2045-2050; S. D. Smith et al.,1997, J. Urol., 158(3 Pt 1):703-708). Moreover, patients withinterstitial cystitis had improvement in urinary symptoms and increasedurinary NO production when treated with oral L-Arginine (M. A. Wheeleret al., 1997, J. Urol., 158(6):2045-2050). Recent evidence has shownthat urethral smooth muscle relaxation is mediated by NO release andthat NO also mediates prostate smooth muscle relaxation (H. Kakizaki etal., 1997, Am. J. Phys., 272:R1647-1656; A. L. Burnett, 1995, Urology,45:1071-1083; M. Takeda et al., 1995, Urology, 45:440-446; W. Bloch etal., 1997, Prostate, 33:1-8).

SUMMARY OF THE INVENTION

It is an object of the present invention to provide new and effectivemethods and compositions for the treatment of various types of diseaseconditions and defects of the musculoskeletal system and the bone, usinggenetically engineered muscle-derived cells, e.g., myoblasts andmuscle-derived stem cells, (also referred to as MDCs herein) in thecell-mediated delivery of exogenous genes for the expression andproduction of encoded gene products. The present invention affords astable gene delivery vehicle to afflicted areas, e.g., the joint(ligament, meniscus, and cartilage), smooth muscle, skeletal muscle andbone, which sustains the production of proteins that amelioratepathological muscle-related conditions, e.g., musculoskeletal and boneconditions. Examples of pathological conditions of the musculoskeletalsystem include arthritis and damage to ligaments, cartilage andmeniscus, resulting in general muscle weakness and/or dysfunction, suchas in the face and hands, as a nonlimiting example. Disease conditionsof the bone include segmental bone fractures, defects, weakness,non-unions and any type of bone augmentation. The present inventionovercomes the problem of transient gene expression, which reduces theefficacy of ex vivo gene transfer to the joint, for example, usingsynovial cells.

Another object of the present invention is to provide a general methodfor muscle-derived cell mediated ex vivo gene transfer involvingharvesting muscle-derived cells, preferably, autologous muscle-derivedcells, culturing the cells, transducing the cultured cells with anappropriate vector in vitro, e.g., a viral vector, harboring at leastone exogenous gene encoding a bioactive molecule, such as a protein,polypeptide, peptide, drug, enzyme, metabolite, hormone and the like,and injecting the transduced muscle-derived cells into or near anaffected area or site of injury, for example, a muscle; a joint,preferably, the knee joint; a bone defect; or a genitourinary tractdefect.

According to the present invention, the method further includesenhancing and/or ameliorating the therapeutic and repair effects of theexpressed bioactive molecule using muscle-derived cell mediated genetransfer to co-deliver gene(s) coding for trophic factors, e.g., growthfactors, or auxiliary proteins and the like, which are also functionallyexpressed to further promote and ameliorate treatment and repair of theaffected tissue. Suitable muscle-derived cells for use are myoblasts.

Also in accordance with the invention, the candidate molecules to bedelivered with the muscle based gene therapy and tissue engineeringinclude bone morphogenetic protein (BMP)-2, (BMP-2), and other subtypesof BMP (e.g. BMP-6 and BMP-12), vascular endothelial growth factor(VEGF), cartilage-derived morphogenetic proteins 1, 2 (CDMP-1, 2) andhedgehog, for the improvement of bone and cartilage healing. Insulinlike growth factor-1 (IGF-1), nerve growth factor and basic fibroblastgrowth factor (bFGF) are used to improve muscle healing followinginjuries. In fact, according to the present invention, the use of theserecombinant human growth factor proteins has been shown to improvemuscle healing following laceration, contusion and strain injuries.

To improve the healing of meniscal injuries, epidermal growth factor(EGF), transforming growth factor α (TGF-α), basic fibroblast growthfactor (bFGF) and platelet derived growth factor A,B (PDGF-A,B) areuseful, since these growth factors are capable of improving meniscalfibrochondrocyte proliferation and increasing the synthesis of collagenand non-collagen proteins.

Finally, the growth factors particularly suited to improve ligamenthealing include platelet derived growth factor (PDGF), transforminggrowth factor-β (TGF-β), and epidermal growth factor (EGF), which arecapable of improving the proliferative capacity of ligament fibroblastand therefore are important candidates to improve ligament healing.

It is another object of the present invention to provide new andeffective methods and compositions for the treatment of various types ofurinary incontinence, particularly stress urinary incontinence, usinggenetically engineered muscle-derived cells in the cell-mediateddelivery of exogenous genes and their encoded gene products to tissuesof the urinary system, such as the urethra and bladder.

It is yet another object of the present invention to provide uniquelyengineered muscle-derived cells for carrying genes encoding products fortreating a number of genetic and pathologic conditions of themusculoskeletal system and for treating and curing various types ofincontinence, as well as for the further enhancement of urinarycontinence. Suitable muscle-derived cells include myoblasts andmuscle-derived stem cells that will eventually differentiate intomyotubes and muscle fibers, as well as into other lineages such asosteoblasts, chondrocytes, and smooth muscle cells, in particular, whenmuscle-derived stem cells are used.

Another object of the present invention is to inject autologousmuscle-derived cells (e.g., myoblasts, and muscle-derived stem cells(MDCs)) that have been transfected or transduced with a vector (e.g.,viral and non-viral) containing at least one gene encoding a bioactivemolecule and, optionally, at least one gene encoding a trophic factor,e.g., a growth factor or a neurotropic factor, into a muscle tissue,e.g., the urethral wall as an effective treatment for stress urinaryincontinence. The muscle-derived cells can be cultured and harvested andcan generate sufficient quantities of muscle cells for repeatedinjections. The present invention is intended to embrace muscle-derivedcells which have been genetically engineered to contain genes encodingboth a bioactive molecule and a trophic factor. Alternatively, differentmuscle-derived cells can be engineered to contain either a gene encodinga bioactive molecule or a gene encoding a trophic factor or an immunesuppression agent. The different muscle-derived cells can be co-injectedor injected at different times, or in combination with other transducedmuscle-derived cells, depending upon the type of treatment andtherapeutic enhancement desired.

In accordance with the present invention, muscle-derived cellsexpressing desired gene products comprise a beneficial cell-mediatedgene therapy which allows the survival of injected cells and thepersistence of gene products, including growth factors (e.g. bFGF,IGF-1, VEGF, PDGF A,B, BMP-2, CDMP, etc.) and neurotropic factors (e.g.,nerve growth factor) to treat and improve urinary tract dysfunction overprolonged periods of time.

Yet another object of the present invention is to provide a simpletreatment method for women and men with stress urinary incontinence byusing autologous, transfected muscle-derived cells to enhance theirurinary sphincters. Such muscle-derived cell-mediated gene therapyallows repair and improvement of the urinary sphincter. In accordancewith the present invention the treatment comprises a simple needleaspiration to obtain muscle-derived cells, for example, and a brieffollow-up treatment to inject cultured and prepared cells into thepatient via an outpatient endoscopic procedure. Also according to thepresent invention, autologous muscle cell injections using myoblasts andmuscle-derived stem cells (MDCs) harvested from and cultured for aspecific stress incontinence patient can be employed as a nonallergenicagent to bulk up the urethral wall, thereby enhancing coaptation andimproving the urinary sphincter muscle. In this aspect of the invention,simple autologous muscle cell transplantation is performed, preferablywithout an accompanying gene therapy.

Another object of the present invention is the use of geneticallyengineered muscle-derived cells and cell mediated gene delivery forinjection into the detrusor muscle as a means of modulating bladdercontractility. In accordance with the present invention, survival of themuscle-derived cells and the expression of foreign genes in such cellshave been demonstrated after injection into the bladder wall.Muscle-derived cell mediated gene therapy provides a useful treatmentfor modulating detrusor contractility and for an overactive bladder.

Another object of the present invention is to provide geneticallyengineered muscle-derived cells (e.g., myoblasts and muscle-derived stemcells) carrying the nitric oxide synthase (NOS) gene, preferably,inducible NOS (iNOS), for expression of nitric oxide synthase as atherapy for genitourinary tract dysfunction, for example, male erectiledysfunction, bladder inflammation, or stress incontinence. In accordancewith the present invention, muscle-derived cells carrying the iNOS genehave been demonstrated to successfully deliver the inducible form of NOS(iNOS) into the penis and genitourinary tissue. Moreover, the productionof iNOS, which produces higher quantities of nitric oxide than isproduced by constitutive NOS, by the genetically engineeredmuscle-derived cells stimulated the release of NO and provided asignificant increase in intracavernosal pressure, which was mediated byNO-induced penile vasodilation.

Further objects and advantages afforded by the present invention will beapparent from the detailed description and exemplification hereinbelow.

DESCRIPTION OF THE DRAWINGS

The appended drawings of the figures are presented to further describethe invention and to assist in its understanding through clarificationof its various aspects.

FIGS. 1A-1I present light and fluorescence microscopic analyses ofurethral and neck tissue demonstrating the persistence of injectedmyoblasts carrying the β-galactosidase gene and producingβ-galactosidase, i.e., lacZ, (blue spots) and fluorescent latexmicrospheres (fluorescent green). Increasing magnification (from 40× to100×) of the same specimen is shown, with FIGS. 1A, 1D, and 1G havingthe lowest magnification and FIGS. 1C, 1F, and 1I having the highestmagnification. FIGS. 1A-1C represent bladder neck myoblast injection.FIGS. 1D-1F represent urethral myoblast injection. FIGS. 1G-1I representurethral myoblast injection using a double staining technique in whichboth lacZ staining (blue) and fluorescent latex microsphere labeling(fluorescent green) can be visualized. Many regenerative myofibersexpressing β-galactosidase are seen in the urethral and bladder neckwall. There are large, disorganized patterns of myofibers intermingledwith fluorescent latex microspheres. Hematoxylin-eosin (H and E) tissuestaining was used.

FIG. 2 shows a high magnification (i.e., 100×) of myoblast injectioninto the urethral wall as shown in FIG. 1I. Transduced myoblasts,myotubes and myofibers expressing β-galactosidase (blue color, arrows)are seen in the urethral wall near urethral epithelium (arrow heads). Hand E tissue staining was used.

FIGS. 3A-3F show the results of transducing myoblasts versus synovialcells in vitro (Example 9). Synovial cells (FIGS. 3A, 3B) and myoblasts(FIGS. 3C, 3D) are from cell lines grown in culture. Both cell lineswere infected with an adenovirus vector carrying the LacZ reporter geneusing a similar multiplicity of infection (MOI=25). The expression ofβ-galactosidase by both cell types was observed using LacZhistochemistry at 2 days (FIGS. 3A, 3C) and 6 days (FIGS. 3B, 3D)post-infection. The transduced myoblasts were shown to preserve theirability to differentiate into myotubes expressing β-galactosidase (FIG.3D). Desmin immunofluorescence of myoblast cultures indicated thepresence of multiple, long, pluri-nucleated myotubes where the myoblastswere allowed to differentiate using fusion media (FIG. 3E). The amountof β-galactosidase production by the four different cell cultures at twodays post-infection was quantified using the lacZ assay. Theimmortalized myoblasts produced nearly 5 times more β-galactosidase thandid primary myoblasts, primary synovial cells and immortalized synovialcells. Magnifications A-E: 10×.

FIG. 4 shows interleukin-1 receptor agonist protein (IRAP) production(ng/ml/10⁶ cells as measured by ELISA) after either synovial cells (syn)or myoblasts (myo) transduced with adenoviral vector carrying the geneencoding IRAP (ad-IRAP) were used to infect rabbit joint.

FIGS. 5A-5D show the results of myoblast-mediated ex vivo gene transferinto rabbit meniscus. Myoblasts transduced with an adenovirus vectorcarrying the gene encoding β-galactosidase (LacZ) were injected intorabbit meniscus. FIGS. 5A and 5B show the expression of LacZ in themeniscus following injection and expression of β-galactosidase. FIG. 5Cshows that LacZ staining is co-localized with fluorescent latexmicrospheres in the injected area. FIG. 5D shows the expression ofdesmin, a myogenic marker (green fluorescence) showing the presence ofmuscle cells in the meniscus.

FIGS. 6A-6D show the results of myoblast-mediated ex vivo gene transferinto rabbit ligament. Myoblasts transduced with an adenovirus vectorcarrying the gene encoding β-galactosidase (LacZ) were injected intorabbit ligament. FIGS. 6A and 6B show the expression of LacZ in theligament following injection and expression of β-galactosidase. FIG. 6Cshows that LacZ staining is co-localized with fluorescent latexmicrospheres in the injected area. FIG. 6D shows the expression ofdesmin, a myogenic marker (green fluorescence) showing the presence ofmuscle cells in the ligament.

FIGS. 7A-7H depict the characterization of the survival of differentpopulations of muscle-derived cells following transplantation inskeletal muscle. The injection of the muscle-derived cells obtainedfollowing preplate #1 was rapidly lost by 48 hours post-injection (FIG.7A): only 17% of the LacZ transgene expression present in the injectedmyoblasts pre-injection was measured in the injected muscle. The cellsisolated at preplate #2 (FIG. 7E) led to 55% myoblast loss; preplate #3(FIG. 7B) a 12% loss; and preplate #6 (FIG. 7F) a 124% gain in the levelof transgene expression present in the cells before transplantation. A96% loss of the pure population of myoblasts isolated from myofibers wasobserved at 48 hours post-transplantation (Fiber myoblast, FMb, FIG.7C). Similarly, the immortalized mdx myoblast cell line showed cell lossfollowing transplantation: 93% of the level of transgene expressionpresent in the cell culture post-implantation was seen 2 dayspost-injection (Mdx cell line, FIG. 7G). PP#3 and PP#6 (FIGS. 7D and 7H)displayed a better cell survival at 2 days post-injection, yet adecrease was observed in the amount of LacZ reporter gene in theinjected muscle at 5 days post-injection. However, the cells whichdisplayed a better survival (PP#3 and PP#6) remained with a higher levelof gene transfer at 5 days post-injection. “*” indicates a significantdifference (P<0.05) when compared with transduced non-injected myoblasts(0 hour).

FIGS. 8A-8D shows the ability of engineered myoblasts expressinganti-inflammatory substance IRAP to circumvent the poor survival of theinjected cells. The survival of the myoblasts engineered to expressinterleukin-1 receptor antagonist protein (IL-1Ra) (FIG. 8B) wascompared with the non-engineered control cells (FIG. 8A). Thenon-engineered cells were rapidly lost by 48 hours post-injection(Control myoblast). In contrast, the cells engineered to express IL-1Rasignificantly reduced the early loss of the injected cells (IL-1Raexpressing myoblast): only 20% of the injected cells were lost at 48hours post-injection. However, a significant reduction in the amount ofβ-galactosidase expression was observed at 24 hours post-injectioncompared with the non-injected myoblasts. A high number of transducedmyofibers persisted between day 2 and day 5 following injection (FIGS.8C, 8D). The absence of a significant difference for both populations ofcells at 0 and 0.5 hours post-injection suggested that the loss ofmyoblasts was minimal during injection. “*” indicates a significantdifference (P<0.05) compared with transduced non-injected myoblasts (0hour).

FIG. 9 presents the levels of alkaline phosphatase activity (ALP), U/L,after various muscle-derived cell populations (pp1-pp6) are stimulatedwith osteogenic protein BMP-2, t=30 minutes. The cell types are stromalcells (control) and preplated (pp) cells #1, 2, 3, 5 and 6 as described.The PP#6 corresponds to BMP-2 by producing alkaline phosphatase in adose dependent manner and at a level similar to that observed withstromal cells (SC)

FIG. 10 presents the percentage of desmin-positive cells followingdifferent numbers of doses (100 ng/ml) of BMP-2. It was observed thatstimulation of BMP-2 not only increased the level of alkalinephosphatase expression by the muscle-derived cells, but also decreasedthe number of desmin positive cells in the population of muscle-derivedcells.

FIG. 11 shows that injected muscle-derived cells (PP#6) stimulated withBMP-2 and inserted into a theracyte immunoisolation device (described inExample 1) which was implanted subcutaneously are capable ofparticipating in bone formation as seen by von Kossa (mineralization)and hematoxylin/eosin. These results suggest that muscle-derived cellsare capable of forming bone.

FIG. 12 shows a schematic representation of the construction of ashuttle plasmid to construct an adeno-associated virus to carry theexpression of IGF-1 (muscle injuries), VEGF (bone and cartilagehealing), and BMP-2 (bone and cartilage healing). This shuttle plasmid,designated pXX-UF1, is used to construct an adeno-associated virus.

FIG. 13 shows the results of the use of a muscle biopsy for cartilagehealing. In the frames as shown, the muscle biopsy is seen to beencasing the cartilage defect at 3 weeks post-injection and muscle andcartilage formation is apparent. The muscle biopsy can therefore be usedas a biological scaffold to deliver growth factors, as well as a sourceof pluripotent muscle-derived cells to improve the healing of cartilagedefect. In this figure, “M” represents muscle, while “C” representscartilage.

FIGS. 14A-14C show the results of myoblast-mediated ex vivo genetransfer into rabbit cartilage. Myoblasts transduced with an adenovirusvector carrying the gene encoding β-galactosidase (LacZ) were injectedinto rabbit cartilage. FIGS. 14A and 14B show the expression of desmin,a myogenic marker (green fluorescence), which reveals the presence ofmuscle cells in the cartilage. FIG. 14C shows the expression ofβ-galactosidase in the injected cartilage with myoblasts transduced withadenovirus carrying the expression of β-galactosidase.

FIGS. 15A-15C show the results of primary muscle-derived cell injectioninto the lower urinary tract. (Example 4) Myoblasts transduced with anadenovirus vector carrying the gene encoding β-galactosidase (LacZ) wereinjected into mouse bladder and urethra. FIG. 15A shows the six-monthpersistence post-injection without damage to the bladder wall. FIG. 15Bshows the assays for β-galactosidase in the injected bladder ismaintained approximately 66% after 70 days. FIG. 15C shows the crosssection of a rat urethra. Injection of primary rat muscle-derived cellsresulted in a large bulking effect in the urethra wall.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides genetically engineered muscle-derivedcells containing at least one heterologous nucleic acid (i.e., exogenousto the muscle cells) encoding a desired gene product, such as a protein,polypeptide, peptide, hormone, metabolite, enzyme, or a trophic factor,including cytokines, in which the gene product(s) is/are expressed in asustained fashion in the cells, and are delivered therapeutically by theengineered cells to a tissue or organ site to promote healing afterinjury, or to remedy a localized organ or tissue dysfunction. Tissuesand organs suitable for muscle-derived cell-mediated gene deliveryaccording to the present invention include the musculoskeletal system(e.g., joint), bone, and urogenital system (e.g., urethra, bladder,sphincter).

More particularly, the present invention provides such geneticallyengineered muscle-derived cells (MDCs), e.g., myoblasts, to improve andexpand the treatment of several types of bladder dysfunction includingimpaired bladder contractility. Also, the present invention provides forthe first time the use of skeletal muscle cells for the repair ofurinary tract smooth muscle dysfunction.

The present invention further provides a revolutionary new treatment forurinary incontinence caused by urethral and bladder impairment ordysfunction. Men and women afflicted with stress incontinence aretreated by using autologous muscle-derived cell injection (i.e., such asmyoblasts, harvested from the patient) to build up and support theurinary sphincter. The present invention relates to muscle-derived cellsinjected into the bladder wall as a cellular myoplasty technique toimprove detrusor contractility and relates to muscle-derivedcell-mediated expression of nitric oxide synthase (NOS) as gene therapyfor the treatment of lower urinary tract dysfunction.

A number of muscle-derived or myogenic cells are suitable for use in thepresent invention. Nonlimiting examples of such cells include myoblasts,fibroblasts, adipocytes and muscle-derived stem cells which reside inmuscle tissue. Also intended for use in the present invention areskeletal myoblasts from skeletal muscle, particularly for use in therepair of smooth muscle dysfunction in the urinary tract. In thepractice of the present invention, muscle-derived cells are capable ofdelivering genes not only to skeletal and smooth muscle, but also tobone, cartilage, ligaments and meniscus.

In accordance with the present invention, muscle-derived cells,including myoblasts, may be primary cells, cultured cells, or cloned.They may be histocompatible (autologous) or nonhistocompatible(allogeneic) to the recipient, including humans. Such cells aregenetically engineered to carry specific genes encoding particular geneproducts and/or drug products, and can serve as long-term local deliverysystems for a variety of treatments, for example, for the treatment ofsuch diseases and pathologies as bladder cancer, transplant rejection,neurogenic bladder conditions, e.g., those secondary to diabetesmellitus, and for the regeneration of muscle and nerve.

Preferred in the present invention are myoblasts and muscle-derived stemcells, and more preferred are autologous myoblasts and muscle-derivedstem cells which will not be recognized as foreign to the recipient. Inthis regard, the myoblasts used for cell-mediated gene transfer ordelivery will desirably be matched vis-á-vis the majorhistocompatibility locus (MHC or HLA in humans). Such MHC or HLA matchedcells may be autologous. Alternatively, the cells may be from a personhaving the same or a similar MHC or HLA antigen profile. The patient mayalso be tolerized to the allogeneic MHC antigens. The present inventionalso encompasses the use of cells lacking MHC Class I and/or IIantigens, such as described in U.S. Pat. No. 5,538,722.

Myoblasts, the mononucleated muscle cells, are uniquely different fromother cells in the body in a number of ways: 1) myoblasts naturallydifferentiate to form muscle tubules capable of muscle contraction, 2)when myoblasts fuse to form myotubes, these cells become post mitotic(stop dividing) with maturation, thus allowing control of the number andamount of myoblasts per injection, and 3) as myotubes, the cells expresslarge amounts of protein which is produced in the cells due tomultinucleation.

In accordance with the present invention, muscle-derived cells,including myoblasts, may be genetically engineered by a variety ofmolecular techniques and methods known to those having skill in the art,for example, transfection, infection, or transduction. Transduction asused herein refers to cells which have been genetically engineered tocontain a foreign or heterologous gene via the introduction of a viralvector into the cells. Muscle-derived cells, including myoblasts, can betransduced by different viral vectors and thus can serve as genedelivery vehicles to transfer expressed proteins into muscle.

Although viral vectors are preferred, those having skill in the art willappreciate that the genetic engineering of cells to contain nucleic acidsequences encoding desired proteins or polypeptides, cytokines, and thelike, may be carried out by methods known in the art, for example, asdescribed in U.S. Pat. No. 5,538,722, including fusion, transfection,lipofection mediated by the use of liposomes, electroporation,precipitation with DEAE-Dextran or calcium phosphate, particlebombardment (biolistics) with nucleic acid-coated particles (e.g., goldparticles), microinjection, and the like.

The present invention also relates to vehicles or vector constructs forintroducing heterologous (i.e., foreign) nucleic acid (DNA or RNA), or asegment of nucleic acid that encodes a functional bioactive product,into muscle-derived cells, in which the vectors comprise a nucleic acidsequence read in the correct phase for expression. Such vectors orvehicles will, of course, possess a promoter sequence, advantageouslyplaced upstream of the sequence to be expressed. The vectors may alsocontain, optionally, one or more expressible marker genes for expressionas an indication of successful transfection and expression of thenucleic acid sequences contained in the vector. To insure expression,the vectors contain a promoter sequence for binding of the appropriatecellular RNA polymerase, which will depend on the cell into which thevector has been introduced. For example, the promoter for expression inmuscle-derived cells, such as myoblasts, is a promoter sequence to whichthe cellular RNA polymerases will bind.

Illustrative examples of vehicles or vector constructs for transfectionor infection of muscle-derived cells include replication-defective viralvectors, DNA virus or RNA virus (retrovirus) vectors, such asadenovirus, herpes simplex virus and adeno-associated viral vectors.Adeno-associated virus vectors are single stranded and allow theefficient delivery of multiple copies of nucleic acid to the cell'snucleus. Preferred are adenovirus vectors. The vectors will normally besubstantially free of any prokaryotic DNA and may comprise a number ofdifferent functional nucleic acid sequences. An example of suchfunctional sequences may be a DNA region comprising transcriptional andtranslational initiation and termination regulatory sequences, includingpromoters (e.g., strong promoters, inducible promoters, and the like)and enhancers which are active in muscle cells. Also included as part ofthe functional sequences is an open reading frame encoding a protein ofinterest, and may also comprise flanking sequences for site-directedintegration. As a particular example, in some situations, the5′-flanking sequence will allow homologous recombination, thus changingthe nature of the transcriptional initiation region, so as to providefor inducible or noninducible transcription to increase or decrease thelevel of transcription, as an example.

In general, the nucleic acid desired to be expressed by themuscle-derived cell is that of a structural gene, or a functionalfragment, segment or portion of the gene, that is heterologous to themuscle-derived cell and encodes a desired protein or polypeptideproduct, for example. The encoded and expressed product may beintracellular, i.e., retained in the cytoplasm, nucleus, or an organelleof a cell, or may be secreted by the cell. For secretion, the naturalsignal sequence present in the structural gene may be retained, or asignal sequence that is not naturally present in the structural gene maybe used. When the polypeptide or peptide is a fragment of a protein thatis larger, a signal sequence may be provided so that, upon secretion andprocessing at the processing site, the desired protein will have thenatural sequence. More specific examples of genes of interest for use inaccordance with the present invention include the genes encoding nitricoxide synthase; trophic factors, including growth factors and cytokines,such as basic and acidic fibroblast growth factors (bFGF and aFGF),nerve growth factor (NGF), brain derived neurotrophic factor (BDNF),neurotrophin, insulin-like growth factor (IGF), transforming growthfactor alpha (TGF-α), transforming growth factor beta (TGF-β), plateletderived growth factor (PDGF) and the like; hormones; metabolic products,generally of low molecular weight; diffusable products; serum proteins;osteogenic proteins, e.g., BMP-2.

As mentioned above, a marker may be present for selection of cellscontaining the vector construct. The marker may be an inducible ornon-inducible gene and will generally allow for positive selection underinduction, or without induction, respectively. Examples of marker genesinclude neomycin, dyhydrofolate reductase, LacZ, and the like.

The vector employed will generally also include an origin of replicationand other genes that are necessary for replication in the host cells, asroutinely employed by those having skill in the art. As an example, thereplication system comprising the origin of replication and any proteinsassociated with replication encoded by a particular virus may beincluded as part of the construct. As a caveat, the replication systemmust be selected so that the genes encoding products necessary forreplication do not ultimately transform the muscle-derived cells. Suchreplication systems are represented by replication-defective adenovirusconstructed as described by G. Acsadi et al., 1994, Human Mol. Genetics,3(4):579-584, and by Epstein-Barr virus. Examples of replicationdefective vectors, particularly, retroviral vectors that are replicationdefective, are BAG, described by Price et al., 1987, Proc. Natl. Acad.Sci., 84:156; and Sanes et al., 1986, EMBO J., 5:3133. It will beunderstood that the final gene construct may contain one or more genesof interest, for example, a gene encoding a bioactive metabolicmolecule, e.g., NOS, iNOS, or NO, and a gene encoding a cytokine, e.g.,bFGF, along with the sequences allowing for the proper expression andproduction of the gene products by the engineered cells. In addition,cDNA, synthetically produced DNA or chromosomal DNA may be employedutilizing methods and protocols known and practiced by those havingskill in the art.

If desired, infectious replication-defective viral vectors may be usedto genetically engineer the cells prior to in vivo injection of thecells. In this regard, the vectors may be introduced into retroviralproducer cells for amphotrophic packaging. The natural expansion ofmuscle-derived cells, such as myoblasts, into adjacent regions obviatesa large number of injections into the muscle fibers at the site(s) ofinterest.

In one embodiment of the present invention muscle-derived cells aretransduced with nucleic acid encoding a particular gene product, e.g., agene encoding inducible nitric oxide synthase (iNOS). The transducedmuscle-derived cells, which contain, express and produce the iNOSproduct, are used in cell-mediated transplantation or gene therapytechniques for the treatment of genitourinary tract dysfunction.Examples of lower urinary tract dysfunction include, but are not limitedto, erectile dysfunction of the penis, pyronies disease of the penis;dysfunctions of the urethra, such as stress urinary incontinence,bladder outlet obstruction, urethritis, dysfunction voider; bladderdysfunctions, such as impaired bladder contractility, neurogenicbladder, cystitis and bladder inflammatory disease; and dysfunction ofthe female sexual and reproductive organs, such as vagina, cervix,uterus, fallopian tubes and ovaries.

The same or different muscle-derived cells may also be co-transducedwith heterologous nucleic acid encoding trophic factors whose expressionin and production by the muscle-derived cells aid in effecting and/orenhancing the therapeutic uses of the transduced muscle-derived cells incell-mediated gene therapy. Trophic factors such as cytokines arepreferably used. More specifically, useful cytokines include thosepresented hereinabove, among which are basic fibroblast growth factor(bFGF), nerve growth factor (NGF) and interleukins, such as IL-1 andIL-6.

Muscle-derived cells engineered to contain nucleic acid encoding one ormore trophic factors can be administered as a treatment at the same timeas muscle-derived cells containing nucleic acid encoding a therapeuticprotein or a bioactive molecule, such as a protein, polypeptide,peptide, hormone, metabolite, drug, enzyme, and the like. Alternatively,muscle-derived cells engineered to express trophic factors may beadministered at a later or earlier time, depending on the type oftreatment desired.

In general, an injection of genetically engineered muscle-derived cells,including myoblasts and muscle-derived stem cells, into a given tissueor site of injury comprises a therapeutically effective amount of cellsin solution or suspension, preferably, about 10⁵ to 10⁶ cells per cm³ oftissue to be treated, in a physiologically acceptable medium, such assaline or phosphate buffered saline, and the like.

In a preferred aspect, the present invention provides ex vivo genedelivery to cells and tissues of a recipient mammalian host, includinghumans, through the use of muscle-derived cells, e.g., myoblasts, thathave been virally transduced using an adenoviral vector engineered tocontain a heterologous gene encoding a desired gene product. Such an exvivo approach provides the advantage of efficient viral gene transferwhich, in cases of treatment of muscle-related dysfunction and defectsas described herein, is superior to direct gene transfer approaches. Theex vivo procedure involves the establishment of a primary muscle-derivedcell culture from isolated cells of muscle tissue. The muscle biopsythat will serve as the source of muscle-derived cells can be obtainedfrom the injury site or from another area that may be more easilyobtainable from the clinical surgeon.

The muscle-derived cells are first infected with engineered viralvectors containing at least one heterologous gene encoding a desiredgene product, and then are injected into the same host. In the case ofmyoblasts as an example, the injected, transduced, isogenicmuscle-derived cells then fuse to form myotubes at and near the site ofinjection. The desired gene product is expressed by the injected cellswhich thus introduce the gene product into the injected tissue, e.g.,muscle. The introduced gene products can promote and enhance muscleregeneration and muscle strength in vivo to ameliorate muscle healingfollowing injuries.

In a particular embodiment of the present invention, muscle-derived cellinjection, preferably autologous myoblast injection, into the urethralwall is employed as a treatment for stress urinary incontinence toenhance, improve, and/or repair the urinary sphincter. Muscle-derivedcells, preferably myoblasts, carrying one or more transduced ortransfected heterologous nucleic acids encoding a bioactive moleculeand/or a trophic factor, are injected into the urethral wall and surviveand differentiate into myofibers to improve sphincter function. Thefeasibility and survival of myoblast injection into the urethral wall isdemonstrated in Example 2. In accordance with this embodiment,autologous muscle-derived cell injections (i.e., muscle-derived cellsharvested from and cultured for a specific stress incontinence patient)can be used as a nonallergenic agent to bulk up the urethral wall,thereby enhancing coaptation and improving the urinary sphincter muscle.

In another embodiment of the present invention, muscle-derived cells areinjected into the bladder wall to improve detrusor contractility.Muscle-derived cells, such as myoblasts and muscle-derived stem cells,injected into the bladder wall are capable of surviving anddifferentiating into myofibers that can augment detrusor contractilityas demonstrated in Example 3. In addition, muscle-derived cells whichhave been genetically engineered to carry a foreign gene express theforeign gene product after injection into the bladder wall.

In accordance with the present invention, autologous muscle-derivedcells administered directly into the bladder and urethra exhibitlong-term survival. The use of cell-mediated gene therapy involvinggenetically engineered muscle-derived cells is advantageous over the useof other forms of gene therapy, i.e., gene therapies involving thedirect administration of virus or plasmid vectors, for example. As anexample, the type of muscle-derived cell may contribute to the survivalof the injected cells post-transplantation. In this regard, autologousprimary myoblasts can be harvested and cultured myoblasts can be storedand used in sufficient quantities for repeated urethral and bladderinjections. Autologous myoblast injection results in safe andnonimmunogenic long-term survival of myofibers in the lower urinarytract. (see Example 4 and FIGS. 15A-15C).

In a particular embodiment, muscle-derived cell mediated gene therapy ofthe present invention further involves muscle-derived cells, e.g.,myoblasts, transduced with an adenovirus vector carrying the bFGF gene,thereby allowing expression of bFGF by the transduced muscle-derivedcells in a given tissue. bFGF engineered muscle-derived cells areinjected into the urethra to treat stress incontinence and also into thebladder wall to improve detrusor contractility. In accordance with thepresent invention, injection of bFGF engineered muscle-derived cellsallows improvement in survival and function versus non-engineeredmuscle-derived cells. Following short-term experiments, long-termexperiments were conducted using autologous primary myoblast-bFGFinjection into the bladder and urethra at 4, 14, and 30 days, asdescribed in Example 6. Cell mediated gene therapy using transducedmyoblasts which secreted the trophic factor bFGF provided furtherimproved success in overcoming dysfunctions in the urethra and bladdercompared with non-bFGF-secreting myoblasts.

In another preferred embodiment, adenoviral vectors carrying theinducible nitric oxide synthase (iNOS) gene are introduced intomuscle-derived cells and the resulting transduced cells are used incell-mediated gene therapy. When such transduced muscle-derived cellsare administered locally to the urethra and bladder, dramatic functionalmodifications are demonstrated, e.g., decrease in bladder inflammationand improvement in urethral relaxation (see Example 7). In this aspectof the present invention, iNOS engineered muscle-derived cell-mediatedgene therapy in the bladder provided a diminution of bladderinflammation. In addition, iNOS engineered myoblast injection into theurethra was demonstrated to be useful to decrease urethral outletobstruction. According to the present invention, myoblast mediated iNOSgene therapy resulted in increased local NO production in injectedtissue. iNOS gene therapy in the bladder also decreased thecyclophosphamide (CYP)-induced bladder inflammatory response. iNOS genetherapy into the urethra further induced sustained urethral smoothmuscle relaxation as described in Example 7.

In another embodiment of the present invention, the use ofmuscle-derived cell-mediated gene transfer to the musculoskeletalsystem, such as the joint, offers numerous advantages. A muscle biopsyfor the isolation of muscle-derived cells, e.g., myoblasts, for use asgene delivery vehicles in accordance with the present invention is mucheasier and less invasive than, for example, surgical synovial capsulebiopsy for the isolation of synovial cells. In a muscle biopsy, a smallarea of muscle tissue generally contains enough myogenic cells toproduce quickly millions of muscle-derived cells in culture. Formyoblasts, once the cells are isolated and grown in culture, it is easyto distinguish pure myoblasts from other cell types, since myoblastsfuse to form elongated myotubes in vitro. In addition, desmin, amyogenic specific marker protein, can be used to determine themyogenicity index of the cell culture without the requirement ofdifferentiation. In contrast, because synovial cells are more difficultto distinguish from other cell types, obtaining a pure synovial cellculture is problematic for the treatment of muscle disease ordysfunction.

In addition, before fusion, myoblasts are over five times moreefficiently transduced than synovial cells using the same number ofadenoviral particles per cell in vitro. The differentiation oftransduced myoblasts into myotubes increases the level of geneexpression in the differentiated myotubes and myofibers. One of themajor advantages of myoblasts over other cell types, such as synovialcells, is the myoblast's ability to fuse and become a differentiatedpost-mitotic cell, which can persistently express a high level of anexogenous gene product. In fact, myoblast mediated gene expression haspersisted for at least 35 days in the joint. This allows for thepersistent and efficient expression and production of any proteins ofinterest in the joint.

In addition, myoblasts transduced with adenoviral vectors carrying thegene encoding interleukin-1 receptor agonist protein (IRAP) producednine times more IRAP as measured by ELISA than infection by similarlytransduced synovial cells. The results indicate that myoblasts areintroducing more protein following gene transfer into the knee jointsynovial lining adjacent to the patella. (FIG. 4).

Moreover, myoblasts have been demonstrated to survive in newborn andadult articular joints (knee) of animal models. Such myoblasts haveadhered to most of the structures of a newborn knee, including patellarligaments, cruciate ligaments, meniscus, synovium and joint capsule. Bycontrast, synovial cells have survived in the knee by adhering only tothe synovial lining.

In addition, injections of muscle-derived cells directly into theintra-articular structures, including ligament and meniscus, results ina higher gene transfer than that observed using injection into the jointfluid. By the practice of the present invention, it has been shown thatmyoblasts engineered with an adenovirus carrying the expression of theβ-galactosidase reporter gene are capable of delivering a higherefficiency and a long term persistence of the reporter gene whencompared to the use of ligament fibroblasts and direct adenoviralinjection (FIGS. 6A-6D) Accordingly, adenovirally transduced myoblastswere able to deliver genes and express gene products in the ligament andmeniscus of rabbit at 1 week post injection (FIGS. 5A-5D, meniscus andFIG. 6A-6D, ligament), thereby indicating that that the use of thistechnology can help to improve ligament healing following injuries.Thus, as described herein, injection directly into the intra-articularstructures or into joint fluid can be used to deliver genes into thejoint.

Stable and persistent gene delivery via muscle-derived cells in thejoints has numerous clinical applications. Genetically engineeredmuscle-derived cells, such as myoblasts and muscle-derived stem cells,can deliver proteins with an anti-arthritic function, such asinterleukin-1 receptor agonist protein (IL-1Ra) or soluble receptors fortumor necrosis factor-α (TNF-α) into an inflamed knee joint (Example 9).This technology can supplant surgical intervention to aid in the healingof different types of damaged tissues (e.g., ligament, meniscus,cartilage) which have poor intrinsic healing capacities in the joint.Also, growth factors can be delivered to the site and persist there inthe practice of the present invention to ameliorate tissue pathologiesin the joint. The high level of gene expression and gene productproduction by the muscle-cell mediated gene transfer technology providesadvantages to the art that were previously unable to be attained usingcurrent methodologies.

In accordance with the present invention, muscle-derived stem cells canbe isolated from skeletal muscle. When appropriately stimulated withbone morphogenetic protein-2 (BMP-2), these cells were capable ofexpressing alkaline phosphatase in a dose dependent manner and, moreimportantly, of actively participating in bone formation in vivo. Thesecells have the ability to differentiate into other lineages.Accordingly, these cells may be used not only to deliver growth factorsand cytokines for the musculoskeletal system, but also to act as anendogenous source of responding cells that may actively participate inthe healing process of the musculoskeletal system.

In another embodiment of the present invention, muscle-derivedcell-mediated gene transfer is employed to treat bone defects (Example10). Because most bone defects are surrounded by muscle, the injectedmuscle-derived cells, such as myoblasts, have a natural myogenic milieuto fuse onto. Muscle biopsies to isolate muscle-derived cells suitablefor use according to the invention are less invasive procedures than arebone marrow biopsies to isolate stromal cells, for example. In fact,most muscle biopsies can be done in an office setting.

In addition, a large percentage of the cells isolated from a skeletalmuscle biopsy are myoblasts, while only 1 to 2% of bone marrow cellshave osteogenic capacity. Myoblasts in cell culture can be furtherpurified using an established technology (T. A. Rando and H. M. Blau,1994, J. Cell. Biol., 125:1275-1287) and muscle cells are relativelyeasy to cultivate in vitro; millions of cells can be grown in a fewdays.

Another advantage of muscle-derived cell mediated gene therapy for thetreatment of bone defects is that muscle cells can transform intoosteoblasts when stimulated by osteogenic proteins, thereby affordingthem osteogenic potential after stimulation. For example, transducedmyoblasts were capable of fusing into myofibers in the bone defect andin the muscles surrounding the defect while expressing a marker gene(Example 10). Thus, in accordance with the present invention,engineering the skeletal muscle cells surrounding the bone defect toexpress or overexpress osteogenic proteins, e.g., BMP-2, allows therelease of these proteins to improve bone healing at the adjacent bonedefect. More importantly, the transplantation of BMP-2 engineeredmuscle-derived cells, which can fuse to form myotubes and myofibersproducing the osteogenic protein in the bone defect, can further enhancebone healing. In fact, muscle-derived cell mediated gene transfer ofosteogenic proteins has the capability of improving both osteoinductiveand osteoconductive aspects of bone healing.

The cyto-architecture of the fused myofibers may not only serve as areservoir of secreting osteogenic proteins, but also as a matrix forbone formation. It is likely that muscle, due to its high vascularity,may improve the revascularization of the bone defect and, therefore, aidin the improvement of bone healing. In accordance with this embodiment,the present invention provides a viable and efficient approach toimproving the healing of both segmental bone defects, bone fractures andnon-unions, and provides cells which can participate in desired boneformation.

In a related aspect of the invention, the present inventors havedetermined that a population of osteoprogenitor cells residing withinskeletal muscle are suitable as vehicles for the delivery of osteogenicproteins such as BMP-2 to the bone defect area. Further, under thestimulation of such osteogenic proteins, the delivery cells themselveshave the potential to become osteoblasts and participate in boneformation (Example 11). Such muscle-derived pre-osteoblastic cells(i.e., pluripotent mesenchymal cells found in skeletal muscle) aresuperior to stromal cells as cell-mediated ex vivo gene deliveryvehicles because of the increased protein capacity of the former cells.

As described in Example 11, a subpopulation of muscle-derived cells,obtainable by an easily reproducible technique, can be induced toacquire alkaline phosphatase activity (ALP) by stimulation with BMP-2.Without BMP-2 stimulation, that same population of cells does not haveALP activity. The ALP activity has a trend toward dose dependence andincreases significantly with additional dosing with BMP-2 over time. Thesame cell population decreases its expression of desmin, a myogenicmarker, with BMP-2 stimulation. The BMP-2 stimulatable, ALP-producingcell population is morphologically distinct from other muscle-derivedcells and divide slowly. Such cells are likely to represent a populationof pluripotent mesenchymal cells present in muscle and useful for cellmediated gene delivery to bone in accordance with the present invention.

EXAMPLES

The examples as set forth herein are meant to exemplify the variousaspects of the present invention and are not intended to limit theinvention in any way.

Example 1

Materials and Methods:

The materials and methods described in Example 1 pertain to thedescription of the invention and the other examples as set forthhereinbelow.

Animals and Myoblast Injection: Adult Sprague Dawley (S.D.) female rats(Hilltop Laboratories, Pittsburgh, Pa.), weighing approximately 250 gwere used in the experiments described. Only certified viral freeanimals were used. The animals were housed in an approved viral genetherapy P-2 facility at the University of Pittsburgh Medical Center.Strict adherence to P-2 protocol was followed. The animals wereanesthetized with pentobarbital (50 mg/kg) for myoblast harvesting,myoblast injection, and during measurement of bladder pressure.

After surgical preparation, a low midline incision was made to exposethe bladder and proximal urethra. During injection using a Hamiltonsyringe, 10-20 microliter (μl) of myoblast suspension (approximately1-2×10⁶ cells per 10 μl) were injected into the bladder or dorsalproximal urethral submucosa of experimental animals (Group 1) in twosites. Identical sites were injected in each animal. Control animals(Group 2) were injected with an equal volume of sterile saline solution.During the first week after the implantation the animals were closelymonitored for any adverse events.

Cryoinjury Model: An 8 mm diameter aluminum rod (Harvard Scientific) wasused as a cryoprobe to cause a full thickness bladder or urethral wallinjury without causing bladder rupture. The cryoprobe was placed in dryice for 1 minute then immediately placed against the bladder wall orurethral wall for 10 seconds without causing bladder rupture asdetermined by histology. This method has been found to cause a reliableand reproducible transmural injury. In sham-treated animals, thecryoprobe was placed against the tissue at room temperature. For bladderinjury, the bladder was filled to 1 ml volume using sterile salinethrough a urethral catheter. Each bladder was injured at one site(bladder dome). Myoblast injection was done 15 minutes after cryo orsham injury at the site of injury. Urethral injury was carried out atthe ventral 12 o'clock position of the proximal urethra with the bladderneck as the cranial margin.

Micturition Pattern Measurement: Urination pattern profiles wereobtained using a specialized metabolic cage which deflects voided urinefor collection and quantification over a 24 hour period. Micturitionparameters evaluated in the described experiments included total urineoutput/24 hours, number of micturitions/24 hours and mean volume permicturition (M. D. Chancellor et al., 1994, J. Urol., 151:250-254; T.Watanabe et al., 1996, J. Urol., 156:1507-1510).

The deflected urine was collected in a cup placed on a strain gaugetransducer (Grass, F3) connected to a strain gauge preamplifier (WordPrecision Instruments, Saratoga, Fla.). The data were input into a dataacquisition system: data acquisition was carried out with an AD card(DATAQ) placed in a Gateway 2000 computer. Appropriate data acquisitionsoftware (i.e., DI-200, DATAQ) and a playback software (i.e., WindaqEx)were installed on the computer.

Cystometrogram (CMG): CMG (i.e., measurement of myoblast urethralinjection; measurement of myoblast bladder injection and measurement ofurethral pressure) was performed under urethane anesthesia (2.4 mg/kg²).Specific techniques of CMG for urethral and bladder injection aredescribed below.

Measurement of Myoblast Urethral Injection: The bladder was cannulatedusing a p-50 catheter placed through the puncture site in the bladderdome and secured with a silk ligature. This catheter was connected to aY connector and to both a Harvard infusion pump and a Grass polygraph.Continuous infusion urodynamic evaluation was conducted with a constantfill rate of 0.075 ml/min. using a Harvard pump. The volume of bladdercapacity, maximum voiding pressure and residual urine volume weredetermined.

Measurement of Myoblast Bladder Injection: The bladder was cannulatedusing a p-50 catheter placed per urethra. This catheter was connected toa Y connector and to both a Harvard infusion pump and a Grass polygraph.Continuous infusion urodynamic evaluation was conducted with a constantfill rate of 0.075 ml/min. using a Harvard pump. The volume of bladdercapacity, maximum voiding pressure and residual urine volume weredetermined. The urethra was cannulated as described in the experiments,rather than the bladder as outlined in the measurement of myoblasturethral injection, so as not to alter bladder function.

Urethral Pressure Measurement: Urethral perfusion pressure andisovolumetric bladder pressure were measured with catheters insertedthrough the bladder dome in urethane anesthetized female S.D. rats. Thepreviously described catheter assembly (H. Kakizaki et al., 1997, Am. J.Physiol., 272:R1647-1656) was wedged in the bladder neck to blockpassage of fluid between the bladder and urethra without affecting thenerve supply to the organs. The external urethra was not catheterized.Responses were examined at an urethral saline infusion speed of 0.075ml/min.

A double lumen transvesical intraurethral perfusion and pressurerecording catheter was prepared as follows: A double lumen bladdercatheter was constructed from PE 200 tubing (outer lumen, free end flameflared) and PE 50 tubing (inner lumen). The PE 200 tube was connected toa syringe for bladder filling and the PE 50 tube was connected to apressure transducer. Both tubes can also be connected, by way ofstopcocks, to a peristaltic pump system set up for isovolumetric fluidexchange as a method for intravesical drug delivery.

A double lumen urethral catheter was constructed from PE 160 tubing(outer lumen) and PE 50 tubing (inner lumen). The PE 160 tubing wasfitted with a pipette tip at one end and was connected to an infusionpump for intraurethral perfusion of either saline or drug solution. ThePE 50 tubing inner lumen extended slightly past the outer lumen, butremained within the pipette tip. The other end was connected to apressure transducer for the measurement of intraurethral perfusionpressure.

The urethral catheter system was passed through the dome of the bladderand was seated snugly in the bladder neck. Both the bladder and urethralcatheters were fixed in place at the bladder dome with suture.

Measurement of the Contraction of the Bladder and Urethral Strips Evokedby Electrical Stimulation: The bladder and urethra were quickly removedfrom the abdomen after decapitation. One longitudinal strip was preparedfrom each bladder and one spiral strip was prepared from the urethra.The preparations were mounted in 5 mL organ baths containing Krebssolution (mmol/L: NaCl 113, KCl 4.7, CaCl₂ 1.25, MgSO₄ 1.2, NaHCO₃ 25,KH₂PO₄ 1.2, glucose 11.5) and constantly bubbled with a mixture of 95%O₂ and 5% CO₂. The initial tension was set to 10 mN and isometriccontractions were measured with strain-gauge transducers coupled with aTBM4 strain gauge amplifier (World Precision Instruments) and recordedon computer using a data acquisition program (Windaq, DATAQ InstrumentsInc, Akron, Ohio, USA). The sampling rate per channel was set to 100 Hz.The amplitude of the stimulation-evoked contractions was computed by acalculation program (WindaqEx, DATAQ). After 20 minutes ofequilibration, electrical field stimuli were applied through twoplatinum wire electrodes positioned on the top and the bottom of theorgan bath separated by 4 cm.

Stimulation Paradigm: The bladder and urethral strips were stimulatedwith square wave pulses of 0.25 msec duration with maximal voltage (100V) and a frequency response curve was constructed using 10 and 80 shocksat 1, 2, 5, 10, 20 and 40 Hz. Subsequently, 5 and 50 μM carbachol wereadded to the bladder preparation to evoke contractions by postsynapticM3 muscarinic receptor activation, and 90 mM K⁺ was applied to directlyactivate the smooth muscle contractile mechanism. In urethralpreparations, 20 Hz/10 shock stimulation was applied to evokecontraction via nerve stimulation. Subsequently, 2 μM phenylephrine wasadded to the bath to check the smooth muscle contractions via thepostsynaptic alpha-1 adrenergic receptor activation. 90 mM K⁺ was thenused to check the smooth muscle responsiveness to direct depolarization.

Bladder Inflammation Model: Rats received a single intraperitonealinjection of CYP (100 mg/kg) (Sigma Chemical) for the bladderinflammation model. Myoblasts transduced by adenovirus carrying the iNOSgene, or sham-treated myoblasts, were injected into the rats at the samesession under brief pentobarbital anesthesia. The sham-treated controlanimals received sterile saline injection. Animals were monitored formicturition pattern for 4 days before CMG studies and sacrifice. Bladdertissue was harvested for contractility studies and then processed forimmunohistochemical studies.

Urethral Obstruction Model: A modification of the technique described byB. Uvelius and A. Mattiasson, 1984, J. Urol., 132:587-590 and W. D.Steers et al., 1991, J. Urol., 155:379-385 was used to create partialbladder outlet obstruction.

Briefly, female S.D. rats (approximately 250 grams) underwent a partialsuture ligation of the proximal urethra under a brief pentobarbitalanesthesia through a small midline incision. The urethral diameter wasreduced to 1 mm by tying two 4-0 nylon sutures around the urethra and anextraluminally-positioned (1 mm O.D.) polyethylene tube. The tubing wasthen removed and the abdominal incision was reapproximated. Controlanimals underwent sham surgery in which the urethra wascircumferentially dissected, but not ligated. The animals were closelymonitored for complications and overflow incontinence. Four weeks later,myoblasts transduced with an adenovirus construct harboring the geneencoding human iNOS (myoblast-iNOS), or sham saline, was injected in theobstructed and control animals (approximately 8 animals in each of 4groups). Four days later, the animals were monitored for micturitionpattern before CMG studies and sacrifice. Micturition pattern was alsoperformed for 24 hours immediately prior to myoblast injection. Urethraland bladder tissue were harvested for contractility studies and thenprocessed for immunohistochemical studies and PCR.

Purification of Primary Myoblasts: The forelimbs and the hindlimbs wereremoved from neonatal mice (T. A. Rando et al., 1994, J. Cell Biol.,125:1275-1287; Z. Qu et al., 1998, J. Cell Biol., 142:1257-1267) and thebone was dissected. The remaining muscle mass was minced into a coarseslurry using razor blades. Muscle cells were enzymatically dissociatedby the addition of collagenase-type X1 (0.2%) for 1 hour at 37° C.,dispase (grade II 240 unit) for 30 minutes and trypsin (0.1%) for 30minutes. The muscle cell extract was pre-plated on collagen-coatedflasks. Different populations of muscle-derived cells were isolatedbased on the number of preplates performed on collagen coated flasks.Preplate #1 (PP#1) represented a population of muscle-derived cells thatadhered in the first hour following isolation; PP#2 represented apopulation of muscle-derived cells that adhered in the next two hours;PP#3 represented a population of muscle-derived cells that adhered inthe next 18 hours; and the subsequent preplates were obtained at 24 hourintervals (PP#4-6). The myogenic population in each flask was evaluatedby desmin staining and on differentiation ability when cultured in afusion medium. The proliferation medium was F10-Ham supplemented with20% fetal bovine serum and 1% penicillin/streptomycin; the fusion mediumwas F10-Ham supplemented with 2% fetal bovine serum and 1% antibioticsolution (penicillin/streptomycin). All the culture medium supplies werepurchased through Gibco, BRL, Grand Island, N.Y., USA. The firstpreplate flasks contained a majority of fibroblasts and the lastpreplate was highly enriched with myogenic cells (desmin positive).

The different populations of cells were infected with β-galactosidaseexpressing adenovirus. The adenovirus, an E1-E3 deleted recombinantadenovirus kindly obtained through GeneVec Inc. (Dr. I. Kovesdi), hadthe β-galactosidase reporter gene under the control of the humancytomegalovirus promoter (HCMV), followed by the SV40 t-intron and apoly-adenylation signal (PolyA). The adenovirally transduced cells werethen transplanted into the hindlimb muscle (gastrocnemius and soleus) ofmdx mice and assayed for their survival post-implantation.

Pure myoblasts were also obtained from isolated viable myofibers asfollows: Immediately after cervical dislocation or decapitation of mdxmice, single muscle fibers were prepared from dissected soleus orextensor digitorum longus (EDL) muscle by enzymatic desegregation in0.2% type 1 collagenase (Sigma, St. Louis, Mo., USA). Isolated musclefibers from 2-day, 15-day, 1-month, 6-week and 6-month-old mice wereused. Due to the small size of 1 to 3-day-old mice, soleus andgastrocnemius were removed en bloc from the lower leg fordisaggregation.

Anterior compartment muscles were prepared by cutting the leg at theankle and knee joints, and adding the entire lower leg to thecollagenase solution. Following isolation of 200 myofibers per muscle, aminimum of 5-10 myofibers per well were plated in 6 well plates coatedwith 1 mg/ml Matrigel (Collaborative Biomedical Products, Bedford,Mass., USA) in 2 ml DMEM medium containing 10% horse serum and 1% chickembryo extract, 2% L-glutamine and 1% penicillin/streptomycin (SigmaCo.). Plates were placed in a 37° C., 5% CO₂ incubator for several days.The cells emerging from the cultured myofibers were grown untilconfluent, assayed for desmin expression, transduced with adenoviruscarrying LacZ reporter gene expression and tested for myoblast survivalafter implantation according to the procedure described herein.

An immortalized mdx cell line was also used. This cell line was isolatedfrom a transgenic animal carrying a thermolabile SV40 T antigen underthe control of an inducible promoter as described by J. E. Morgan etal., 1994, Develop. Biol., 162:486-498. The immortalized mdx cell lineproliferated indefinitely under permissive conditions (33° C. with gammainterferon) in proliferation medium (DMEM+20% FBS) and underwent normaldifferentiation at 37-39° C. without gamma interferon in fusion medium(DMEM+2% FBS).

Adenoviral Vectors: Adenoviral vectors carrying the gene encodingβ-galactosidase (i.e., adenovirus-lacZ), the gene encoding bFGF (i.e.,adenovirus-bFGF) and the gene encoding iNOS (i.e., adenovirus-iNOS) asdescribed herein have been constructed. The iNOS gene has been clonedand the vector constructs produced as described in D. A. Geller et al.,1993, “Molecular cloning and expression of inducible nitric oxidesynthase from human hepatocytes”, Proc. Natl. Acad. Sci. USA,90:3491-3495; in E. Tzeng et al., 1997, “Adenoviral transfer of theinducible nitric oxide synthase gene blocks endothelial cell apoptosis”,Surgery, 122(2):255-263; and in E. Tzeng et al., 1996, “Gene Therapy”(Review), Current Problems in Surgery, 33(12):961-1041. The molecularconstruction of a replication defective adenoviral as employed herein isdescribed in G. Ascadi et al., 1994, Human Mol. Genetics, 3(4):579-584.The technique, which involves construction, propagation, purificationand titration of the first and third generation adenoviral vector, hasbeen described by S. Kochanek et al., 1996, Proc. Natl. Acad. Sci. USA,93:5731-5736 and G. Ascadi et al., 1994, Human Mol. Genetics,3(4):579-584.

Recombinant human iNOS cDNA was cloned from cytokine-stimulated culturedhuman hepatocytes as described in D. A. Geller et al., 1993, Proc. Natl.Acad. Sci. USA, 90:3491-3495. (Complete cDNA sequence GenBank AccessionNo. L09210). The cloned iNOS cDNA was subcloned into the adenovirusvector along with a neomycin-resistance gene to yield the DFGiNOSconstruct. The previously constructed control vector BaglacZ is aretrovirus that expresses the E. coli lacZ gene (encoding β-galactoside)under the transcriptional control of the Moloney murine leukemia viruslong terminal repeat (MOMLV 5′ LTR) and gag pr65 translation initiationcodon, and the neomycin-resistance gene under the control of the SV40early promoter (Price et al., 1987, Proc. Natl. Acad. Sci. USA,84:156-160). Helper-virus-free DFR-iNOS and BaglacZ viral supernatantswere generated from CRIP packaging cells (E. Tzeng et al., 1995, Proc.Natl. Acad. Sci. USA, 92:11771-11775; E. Tzeng et al., 1996, I. Surgery,120:315-321).

Myoblast Preparation: Primary myoblasts were plated at a density of5×10⁴ cells per well in a 6-well plate. After 24 hours, the cells werewashed with Hank's Balanced Salt Solution (HBSS). Sufficient adenovirusstock was aliquotted into each well to achieve a Multiplicity ofInfection equal to 50 (MOI=50). Plates were incubated for 2 hours at 37°C. to allow adequate infection. Proliferation medium (see below) wasthen added to each well. Plates were incubated at 34° C. for 48 hr.Cells can be immunostained for expression of the transgene or harvestedin HBSS for injection into animals. Injections ranged from 1.33×10⁵ to1×10⁶ cell per 100 μl. Tissue was harvested at 48 hours after injection.

Myoblast Transduction with Adenovirus: Primary myoblasts were grown inculture for 5 days in proliferation medium, which contained DMEM and15%-20% fetal bovine serum (Gibco-BRL; NY USA). 6×10⁵ myoblasts wereinfected with adenovirus using a multiplicity of infection (MOI) of 20.Fluorescent latex microspheres (FLMs) were added to the culturedmyoblasts at a dilution of 1:1000 to label the myoblasts, therebyallowing the early fate of the injected myoblasts to be followed in vivo(J. Huard et al., 1994, J. Clin. Invest., 93:586-599; J. Huard et al.,1994, Muscle & Nerve, 17:224-234; J. Huard et al., 1995, Gene Therapy,2:107-115). At 24 hours post-infection the myoblasts were detached usingtrypsin (0.25%) for approximately one minute, centrifuged for fiveminutes at 3500 rpm, and the myoblast pellet was reconstituted with 25μl of HBSS (Gibco-BRL, NY USA). The harvested cells were transplantedusing the technique described below.

Tissue Harvest and Histology: Tissue was harvested at different timesdepending on the experiment. All bladder and urethra specimens werefrozen or fixed in 10% buffered formalin depending on the requirement ofthe assay. The area around each injection site was examinedmicroscopically, stained for LacZ reporter gene andimmunohistochemistry, and photographed. Appropriate assays for the LacZreporter gene and fibroblast growth factor gene expression were carriedout.

LacZ Staining by Histochemical Technique: The cryostat sections of theinjected and control tissues were stained for LacZ expression asfollows. Sections were first fixed with 1.5% glutaraldehyde (Sigma) forone minute, rinsed twice in phosphate-buffered saline (PBS) and thenincubated in X-gal substrate [0.4 mg/ml5-bromo-chloro-3-indolyl-β-D-galactoside (Boehringer-Mannheim,Indianapolis Ind., USA), 1 mM MgCl₂, 5 mM K₄Fe(CN)₆/5 mm K₃Fe(CN)₆ inPBS)] overnight at 37° C.

Assay for β-Galactosidase Activity: This technique provided betterquantification and comparison of the level of transgene expression ininfected cells as well as in injected tissues (e.g., muscle, bladder,urethra). The injected muscle was frozen in liquid nitrogen andhomogenized in 0.25 M Tris-HCl (pH 7.8) and the homogenates werecentrifuged at 3,500×g for 5 min. The muscle homogenate was disrupted by3 cycles of freeze/thaw and the supernatant was recollected bycentrifugation (12,000×g/5 min. at 4° C.) and transferred to a cleanmicrocentrifuge tube. Thereafter, 30 μl of this extract was mixed with66 μl of 1× o-nitrophenyl-β-D-galactopyranoside; ONPG (4 mg/ml ONPG in0.1 M sodium phosphate pH 7.5), 3 μl of 100×Mg solution (0.1 M. MgCl₂,4.5 M β-mercaptoethanol), 201 μl 0.1 M sodium phosphate (41 ml of 0.2 MNa₂HPO₄. 2H₂O, 9 ml of 0.2 M NaH₂PO₄. 2H₂O and 50 ml H₂O) and thenincubated at 37° C. for 30 minutes, or until a faint yellow color haddeveloped. Finally, the reaction was stopped by adding 500 μl of 1 MNa₂CO₃ and the optical density was read on a spectrophotometer at awavelength of 420 nm. The level of β-galactosidase activity (# ofunits/sample) was extrapolated on a calibration curve which converts theoptical density at 420 nm to the concentration of β-galactosidaseenzyme.

Determination of the Persistence of Myoblast Injection: Myoblasts wereincubated with FLMs and transduced with adenovirus carrying theβ-galactosidase reporter gene. Each flask of 500,000 cells wastrypsinized using 0.5% Trypsin-EDTA, centrifuged at 3500 rpm for 5minutes, and resuspended in 100 μl of medium. The solution from oneflask was injected into the urethra or bladder of the animals. A 25 μlsolution of infected muscle cells (1×10⁶ cells) was injected into theurethra using a Hamilton syringe.

At the various sacrifice intervals, the urethra and bladder wereremoved. After contractility studies, the tissue was snap frozen using2-methylbutane pre-cooled in liquid nitrogen. Analysis of the sectionsincluded hematoxylin-eosin staining; X-gal staining; desminimmunohistochemistry and localization of the FLMs. From these analyses,the location and viability of the injected cells were determined. Inaddition, using different myogenic markers, such as desmin and myosinheavy chains, it could be determined if any myoblast fusion had occurredwith the internal structures of the joint. Desmin and myosin heavychains are assayed to demonstrate that the muscle-derived cells carrythe reporter gene and that reporter protein, e.g., LacZ, is expressed.By co-localizing the LacZ reporter gene with genes encoding musclestructural proteins (e.g., desmin and myosin heavy chains), it could bedetermined that LacZ expression results from the fusion of the injectedcells. Myosin heavy chains are only expressed in muscle cells underdifferentiation (i.e., myotubes and myofibers).

Statistical comparison of the efficiency of myoblast fusion among theprimary cultures was made by counting the number of transduced myoblastsand myofibers in the urethra. In addition, by using the ONPG assay,statistical comparisons of the level of β-galactosidase expressionmediated by injection of transduced myoblasts could be made at each timeinterval. The number of transduced myoblasts and myofibers thatdecreased over time in the injected urethra and bladder was able to bedetermined.

iNOS Immunohistochemistry: Tissue was harvested and fixed as describedfor β-galactosidase staining. Sections were mounted on gelatin-coatedslides. Endogenous peroxidases were inactivated with 0.5% H₂O₂ inabsolute MeOH for 30 minutes at room temperature. Slides were incubatedat room temperature for 1 hour with anti-iNOS (mouse monoclonal,Transduction Laboratory) in normal goat serum (1:50). Biotinylatedanti-mouse (goat, Kirkengaard-Perry) was incubated for 30 minutes atroom temperature, followed by incubation with strepavidin-horseradishperoxidase for 30 minutes. Slides were developed by the addition ofdiaminobenzidine (1 mg/ml)/hydrogen peroxide (0.03%) reagent and allowedto develop for 3 to 5 minutes.

NOS Immunostaining of Myoblasts: Cells were fixed with 4%paraformaldehyde for 20 minutes and were permeabilized with coldmethanol (−20° C.) for 10 minutes. 1% bovine serum albumin in PBS wasused to block cells and dilute reagents. Diluted anti-iNOS antibody(1:250 dilution, rabbit polyclonal, Santa-Cruz) was incubated at roomtemperature for 60 minutes. Biotinylated anti-mouse (goat,Kirkengaard-Perry) was incubated for 30 minutes at room temperature,followed by incubation with strepavidin-cy3 (strepavidin bound to a redfluorescent marker) for 30 minutes. Wells were examined under amicroscope. In addition, anti-adenovirus protein antibody (Santa-Cruz)was selectively used to confirm that the myoblasts were transduced withthe appropriate adenovirus vector.

Nitrite (NO₂ ⁻) Measurement: Cells were passaged to 24 well plates.After 24 hours, the cell medium was replaced with medium alone or mediumcontaining H4B (100 μM; Sigma). Accumulated NO₂ ⁻ in the supernatantswas quantified by employing the Griss reaction (L. C. Green et al.,1982, J. Analyt. Biochem., 126:131-138) using sodium nitrate asstandard.

Determination of Nitric Oxide (NO) Formation: NO formation was measuredand compared in bladder and urethra of iNOS treated versus sham treatedanimals. NO formation was estimated by comparing the tissue content ofits stable oxidation product, nitrite, in the stimulated andunstimulated strips. Tissue nitrite content was measured using apreviously described calorimetric technique (L. J. Ignarro et al., 1987,Biochem. Biophys. Res. Commun., 170:843-850). The weighed tissue sampleswere placed in a glass tube and thawed in 0.5 ml of ice-cold methanol.The tissue was then homogenized using a glass pestle. The homogenate wasallowed to stand for 18 hr at 4° C. to ensure complete extraction ofnitrite. Samples were then placed in a refrigerated centrifuge at 4° C.and centrifuged at 15,000×g for 15 minutes. An aliquot (0.3 ml) of eachsample was mixed with 0.4 ml of 1% sulfanilic acid in 0.4 M HCl. After10 min, 0.3 ml of 1% N-(1-naphthy)-ethylene diamine in methanol wasadded and the absorbance of the resultant pink complex was measured in aspectrophotometer at a wavelength of 548 nm. Nitrite concentration wasinterpolated from a standard line constructed by performing an identicalassay using sodium nitrite standards in the range of 0.5-5 μM, togetherwith methanol blanks.

NOS Activity Assay: NOS activity was measured and compared in bladderand urethra of iNOS treated versus sham treated animals by monitoringthe conversion of [³H]-L-arginine to [³H]-L-citrulline as described byA. L. Burnett, 1995, Urology, 45:1071-1083; D. S. Bredt and S. H.Snyder, 1990, Proc. Natl. Acad. Sci. USA, 87:682-685).

Briefly, tissues were homogenized in ice cold homogenization buffer.Enzyme assays contained 25 μl of tissue supernatant and 100 μl of 1μCi/ml [³H]-L-arginine, 1.2 mM NADPH, and 0.7 mM CaCl₂. After a 15minute incubation at room temperature, the assays were terminated by theaddition of 3 ml of 20 mM Hepes (pH 5.5) with 2 mM EDTA. The entiremixture was applied to 0.5-ml columns of Dowex-50W cation exchange resin(Na+ form) to remove unreacted [³H]-L-arginine. [³H]-L-citrulline in thecolumn eluate was quantified by liquid scintillation spectroscopy. Therecovery rate of [³H]-L-citrulline from the columns was measured for alltissues by preincubating each tissue supernatant with a knownconcentration of [³H]-L-citrulline and then measuring the[³H]-L-citrulline in each column eluate. Column saturation studies werealso done to ensure that all of the [³H]-L-arginine was retained in thecolumn. Additional assays were performed in the presence of excessL-nitroarginine methyl ester (L-NAME), a competitive inhibitor of NOS,to verify the specificity of the assay for the production of[³H]-L-citrulline by NOS catalysis.

Interassay variations were controlled for by standardizing NOS activitymeasurements in tissue against the activity measured in the NOS-rich ratcerebellum which was analyzed in parallel for each assay. The level ofcitrulline will be expressed as picomoles per mg of tissue per minute.

RNA Isolation and Northern Blot Analysis: Total cellular RNA wasisolated from uninfected, BaglacZ and human iNOS treated animals usingRNA-zol B as previously described (P. Chomczynski and N. Sacchi, 1987,Analytical Biochemistry, 162:156-159). Aliquots (20 μg) of RNA wereelectrophoresced on a 0.9% agarose gel and blotted onto GeneScreen(DuPont-NEN, Boston, Mass.). After prehybridization, the membranes werehybridized to a DNA probe (D. A. Geller et al., 1993, Proc. Natl. Acad.Sci. USA, 90:522-526; D. A. Geller et al., 1993, Proc. Natl. Acad. Sci.USA, 90:3491-3495). A 2.3 Kb human iNOS cDNA fragment served as the iNOSprobe, while a 4.1 Kb human endothelial cNOS (ecNOS) cDNA fragmentserved as the ecNOS probe. 18S rRNA served as a control for relative RNAloading.

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) Amplification:Samples were immediately frozen in liquid nitrogen and stored at −80° C.Total cellular RNA was extracted from the samples using the method of P.Chomczynski and N. Sacchi, 1987, Analytical Biochemistry, 162:156-159.Total RNA was subjected to first-strand cDNA synthesis using oligo (dT)primer and MMLV reverse transcriptase (E. S. Kawasaki, PCR Protocols: aguide to methods and applications, Eds. M A Innis, D H Gelfang, J JSninsky, and T J White, Academic Press, New York. pp. 22-27, 1990).Primers were designed to amplify iNOS with the assistance of a PCRprimer design program, PCR Plan (Intelligenetics, Mountain View,Calif.). The sequence of the iNOS 5′ oligonucleotide primer (18 bp) usedwas 5′-AGGACATCCTGCGGCAGC-3′ (SEQ ID NO:1) (E. Tzeng et al., 1996, Mol.Med., 2(2):211-225) spanning from base pair 3426 to 3444 of the humaniNOS cDNA sequence (D. A. Geller et al., 1993, Proc. Natl. Acad. Sci.USA, 90:3491-3495). The sequence of the iNOS 3′ oligonucleotide primer(18 bp) was 5′-GCTTTAACCCCTCCTGTA-3′ (SEQ ID NO:2) (E. Tzeng et al.,1996, Mol. Med., 2(2):211-225) spanning from base pair 3724 to 3741 ofthe human iNOS cDNA sequence (D. A. Geller et al., 1993, Proc. Natl.Acad. Sci. USA, 90:3491-3495). The identity of the amplified cDNAfragment obtained from the RT-PCR reaction with iNOS primers(approximately 316 bp) was confirmed using restriction enzyme analysis.

PCR conditions were as follows: denaturation at 94° C. for 1 min;annealing at 57° C. for 1 min; polymerization at 72° C. for 2 min. PCRreactions were performed in a Perkin Elmer 480 thermocycler usingdifferent numbers of cycles to detect a linear range of input RNA. Theoptimal cycle number was identified as 30 cycles. Rat peritonealmacrophages elicited with thioglycolate and RAW cells (RAW 264.7macrophage cell line) stimulated in vitro with LPS served as positivecontrols for iNOS mRNA. The negative control for each set of PCRreactions contained water instead of DNA template. PCR product (20% ofthe reaction volume) of qualitative RT-PCR was separated byelectrophoresis on a 2% agarose gel and stained with ethidium bromide.

For semiquantitative RT-PCR (E. S. Kawasaki: Amplification of RNA. PCRProtocols: A guide to methods and applications, Eds. M. A. Innis, D. H.Gelfang, J. J. Sninsky and T. J. White, Academic Press, New York. pp.22-27, 1990). ³²P endlabeled 5′ primer was used. Fifteen μl of the PCRreaction was separated on a 10% polyacrylamide gel. After gel drying andexposure to a Phosphorlmager screen (Molecular Dynamics Phosphorlmager,Sunnyvale, Calif.), the relative radioactivity of the bands wasdetermined by volume integration using laser scanning densitometry. Eachgel contained the same positive control which permitted normalization ofsamples and comparison among gels.

In Vivo Extraskeletal Bone Formation Assay: A retroviral vector carryingthe expression of the β-galactosidase gene (rv-lacZ) and adenovirusengineered to express the BMP-2 gene (AdV-BMP-2) were used in theseexperiments. The BMP2-125 plasmid was digested with Sal I, and the 1237bp fragment containing the entire cDNA for human BMP-2 was isolated. TheBMP-2 cDNA was then inserted into the Sal I site of the pAd.lox plasmidsuch that the translation start site was then obtained by cotransfectionof pAd.10× with psi-5 viral DNA into CRE-8 cells as previously describedby S. Hardy et al., 1997, J. Virol., 71:1842-1849).

Primary mdx pp6 cells were transduced with rv-lacZ as a method ofmonitoring the in vivo fate of these cells. 1×10⁶ cells were plated in aT-75 flask. Proliferation medium (F10/HAM media containing 20% fetalbovine serum) was washed off with serial rinses of Hank's Balanced SaltSolution (HBSS, Gibco BRL). Three milliliters of rv-lacZ suspension, ata concentration of approximately 5×10⁵, and 6 μl polybrene diluted with2 ml HBSS, were incubated with the cells for 6 to 8 hours at 37° C.Thereafter, proliferation medium was added to the cells, and the cellswere allowed a recovery period of 24 hours prior to AdV-BMP infection.After the 24 hour period, these cells were again rinsed with HBSS andtransduced with AdV-BMP at a multiplicity of infection (MOI) of 50. Thecells were incubated with the viral suspension for 4-6 hours, at whichtime proliferation medium was added without removal of viral particles.

Prior to injection, cells were rinsed multiple times in HBSS andcounted. Injections of 3.0×10⁵ cells suspended in 20 μl of HBSS wereperformed in severe combined immune deficient (SCID) mice hindlimbmuscle. The cell suspension was then injected with a 30 gauge needle ona gas tight syringe into the exposed triceps surae. Animals weresacrificed at 2, 3, 4, and 5 weeks post-injection.

Ectopic bone formation in the injected limb was analyzed by grossinspection, radiograph, and standard histology. Limbs from sacrificedmice were radiographed with a dental radiography machine. Limbs werethen flash frozen in 2 methylbutane buffer pre-cooled in liquidnitrogen. Frozen samples were sectioned into 10-12 μm sections using acryostat (Fisher Scientific). After fixation with glutaraldehyde (1%),muscle sections were incubated with the X-gal substrate for 1-3 hours toreveal the β-galactosidase activity. Further histological analysisincluded von Kossa, hematoxylin, and eosin staining. All animalprocedures were with the guidelines and approval of Children's Hospitaland the University of Pittsburgh Animal Care Committee according toprotocol 1/98.

Diffusion Chamber Implantations: Primary pp6 cells were transduced invitro with AdV-BMP-2 using an MOI of 50. The cells were trypsinized,counted and 1×10⁶ cells were suspended in proliferation medium. Understerile conditions, the cells were loaded 5 microliter Theracyteimmunoisolation devices (Device Engineering Group, Baxter Healthcare,Round Lake, Ill.) using a gas tight syringe and blunt tipped stainlesssteel needles (J. Brauker et al., 1998, Human Gene Ther., 9:879-888).The loading port was sealed with sterile silicone adhesive (Dow Corning,Midland, Mich.). The Theracyte devices consist of a bilayerpolytetrafluoroethylene (PTFE) membrane with an outer membrane pore sizeof 5 micrometers and an inner membrane pore size of 0.4 micrometers. Thebilayer PTFE membrane precludes host cells from entering the device andimplanted cells from exiting the device. The devices were implantedsubcutaneously in SCID mice under ketamine and xylazine anesthesia. Themice were sacrificed three weeks later using methoxyflurane anesthesiaand cervical dislocation. The devices were flash frozen in2-methylbutane precooled with liquid nitrogen. 10 micrometer thicksections were made using a Cryostat (Microm, HM 505 E) and stained withhematoxylin and eosin, with and without von Kossa's stain

Example 2

Experiments were performed using myoblast injection into the urethralwall as a treatment for stress urinary incontinence.

In these experiments, myoblasts from the GH8 myoblast cell line(available from the American Type Cell Culture depository) transducedwith an adenoviral vector harboring a reporter gene encodingβ-galactosidase (E1-E3 deleted first generation adenovirus), werecultured and injected into the proximal urethral wall of the female S.D.rat.

In addition, the cells were incubated with fluorescent latexmicrospheres (FLM) to follow the fate of the injected cells (see Example1). The transduced, FLM-labeled cells were injected into adult femaleS.D. rats (n=20). A midline incision was made and myoblasts wereinjected into the proximal urethral wall at two sites with a 10 μlHamilton micro syringe. The myoblast concentration ranged from 1.33×10⁵to 1×10⁶. The tissue was harvested 2-4 days after injection and flashfrozen in liquid nitrogen. The tissue was then sectioned, stained withx-gal substrate and then counterstained with Hematoxylin and Eosin (Hand E). Photographs of the slides were taken under light andfluorescence microscopy (FIGS. 1A-1I).

Injection was done in the urethra of control animals and also in animalsthat had urethral injury using a 8 mm diameter aluminum probe cooled indry ice and then placed on the ventral urethra for 10 seconds (seeExample 1). After 2, 4, and 7 days, animals with and without urethralinjury were euthanized and the portion of the urethra containing theengrafted myoblasts, as well as adjacent normal urethra and bladder,were removed and subjected to functional and microscopic studies.Twenty-four hours prior to sacrifice, the animals underwent micturitionpattern analysis in a metabolic cage to determine change in voidingvolume and frequency (see Example 1).

Animals with and without urethras engrafted with myoblasts alsounderwent a 24-hour micturition pattern analysis in a metabolic cageusing methods described by M. B. Chancellor et al., 1994, J. Urol.,151:250-254.

Immediately prior to sacrifice, animals underwent urethral pressureurodynamic study (cystometrogram (CMG)) under urethane anesthesia usingspecially designed urodynamic catheters to allow simultaneous butisolated measurement of bladder and urethral pressure (H. Kakizaki etal., 1997, Am. J. Phy., 272:R1647-1656).

Urethral perfusion pressure and isovolumetric bladder pressure weremeasured with catheters inserted through the bladder dome. Theseparameters were compared in myoblast-injected animals and insham-injected animals, with and without cryoprobe injury. For theseevaluations, specially designed urodynamic catheters were employed toallow simultaneous but isolated measurement of bladder and urethralpressures. The catheter assembly was wedged in the bladder neck to blockpassage of fluid between the bladder and urethra without affecting thenerve supply to the organs, as described in Example 1. The externalurethra was not catheterized. Responses were examined at a urethralsaline infusion speed of 0.075 ml/min.

After the urodynamic experiments, urethral strips were harvested andplaced in Krebs-filled bath chambers to assess contractility. Controland sham-injected animals were assessed. Histochemistry staining forlacZ and fluorescent microscopic analysis were performed to evaluate andquantify the survival and differentiation of myoblasts and to assess theproduction of myotubes. Long-term experiments using autologous primarymyoblast injection described herein into the urethra were carried outfor 30-60 days.

The results of the foregoing analyses are presented in Table 1.

TABLE 1 Urethra Injection of Myoblast vs. Control Control (n = 12)Myoblast (n = 4) Micturition Pattern 24 hr. urine volume (ml) 21.8 +/−3.8 19.8 +/− 4.2 Urine frequency/24 hr   19 +/− 5   11 +/− 3 Meanmicturition  1.0 +/− 0.4  1.8 +/− 0.9* volume (ml) Urethral StudiesBaseline urethral 28.3 +/− 3.1 36.4 +/− 5.5** pressure (mmHg) Bladdercontraction 78.3 +/− 11.2 72.8 +/− 23.3 pressure (mmHg) *(p < 0.01);**(P < 0.5)

These experiments demonstrated a large number of cells in the urethralwall expressing β-galactosidase and containing FLM under fluorescentmicroscopy. Many regenerative myofibers expressing β-galactosidase werealso seen in the urethral wall. Primary myoblasts injected into SCIDmice survived for over 30 days. Animals treated with myoblast injectionwere discovered to have increased (urethral pressure). In animals havingcryoprobe injured urethras, myoblast injection resulted in improvedcontractility.

Example 3

Experiments were performed to demonstrate the feasibility of myoblastinjection into the bladder wall to improve detrusor contractility. Amyoblast cell line transduced with an adenovirus vector carrying aβ-galactosidase reporter gene as described hereinabove was used forthese experiments. The cells were incubated with fluorescent latexmicrospheres (FLM) to follow the fate of the injected cells. Cells wereinjected into adult male and female S.D. rats (n=18). The myoblasts wereinjected into the dome of the bladder and into the right and leftlateral walls near the dome with a 10 μl Hamilton microsyringe. Themyoblast concentration ranged from 1.33×10⁵ to 1×10⁶ cells. The tissuewas harvested after 2-5 days, sectioned and assayed for β-galactosidaseexpression. For gene therapy experiments, myoblasts were transduced withthe adenovirus containing the human inducible nitric oxide synthase(iNOS) gene at a multiplicity of infection of 50 (MOI=50) and wereinjected into the bladder and studied after 2 to 7 days. The results ofthese experiments demonstrated that in the bladder wall there were alarge number of cells expressing B-galactosidase and containing FLM's asdetermined by fluorescence microscopy. Many regenerative myofibersexpressing β-galactosidase were also seen in the bladder wall. Using aporphyrinic microsensor to measure NO, increased basal release of NO (20nM) was detected from the bladder wall area injected with myoblasts thathad been transfected with the iNOS gene.

These studies demonstrated the survival of myoblasts and the expressionof foreign genes in myoblasts following injection into the bladder wall.Thus, myoblast injection and myoblast mediated gene therapy were shownto be useful for modulating detrusor contractility and for counteractingoveractive bladder function.

Bladder or urethral wall cryoinjury was performed in a rat (S.D.) modelas described in Example 1. Two animals each underwent cryoinjury andsham injury with and without myoblast injection. Four days aftercryoinjury and myoblast injection the contractility evaluation wasperformed. Significant histological damage to the bladder and urethralwall were observed after cryoinjury (FIG. 2). The muscle contractilityto electrical field stimulation disappeared after cryoinjury, comparedwith no change in sham injured animals.

Bladder or urethral strips were mounted in organ baths of 5 mL and wereelectrically stimulated with 100V, 0.25 ms at various frequencies. Boththe cryo damaged bladder and urethral strips showed low and nocontractile activity to electrical field stimulation, to indirect smoothmuscle activation via M3 receptor stimulation by carbachol, or to directsmooth muscle activation with high K⁺, compared with the non-damagedcontrols. After myoblast treatment of the cryo-damaged bladder orurethra there was a significant recovery of the contractile activity toelectrical stimulation.

The results are presented in Table 2.

TABLE 2 Micturition Pattern of Bladder Cryoinjury Versus Control Control(n = 12) Cryoinjury (n = 4) 24 hr. urine volume (ml) 21.8 +/− 3.8 19.8+/− 4.2 Urine frequency/24 hr   19 +/− 5   11 +/− 3 Mean micturition 1.0 +/− 0.4  1.8 +/− 0.9* volume (ml) *(p < 0.01)

These experiments clearly demonstrated an alteration of bladder andurethral function with the cryoinjury model.

Example 4

Experiments were performed to assess the long-term survival ofautologous transduced myoblasts in the bladder and urethra.

These experiments were performed in mice having severe combined immunedeficiency, i.e., the SCID mouse model, and subsequently innon-immunodeficient rats. Autologous myoblasts and muscle-derived cellswere harvested and cultured in sufficient quantity for repeated bladderinjections.

The primary myoblast cultures were grown for 3 days in a proliferatingmedium supplemented to contain DMEM and 15% fetal bovine serum(Gibco-BRL). The myoblasts were infected with adenovirus vector.Fluorescent latex microspheres (0.5 μm) were also added to the culturedmyoblasts at a dilution of 1:1000 to allow the fate of the injectedmyoblasts to be followed in vivo (A. Satoh et al., 1993, J. Histochem.Cytochem., 41:1579-1582; S. Floyd et al., 1997, Basic Appl. Myol,7(3&4)). At 48 hours post-infection, the myoblasts were detached fromthe culture container using Trypsin (0.25%), centrifuged for 5 minutesat 3,500 rpm, and the myoblast pellet was reconstituted with 25 ml ofHank's balanced salt solution (HBSS, Gibco-BRL, NY USA).

The transduced myoblast solution was injected into the detrusor wall onthe left side, with the same number of viral particles injected into thecontralateral side of the animal. The injected mice were sacrificed at 4days post-injection. The bladders from the injected animals were removedand snap-frozen in isopentane pre-cooled in liquid nitrogen. The musclewas then cryostat-sectioned in its entirety in a 10 μm thickness andprepared for staining.

The same viral solution was (i) directly injected intramuscularly or(ii) used to transduce myoblasts in vitro prior to the ex vivo genetransfer to the same animal. Newborn (two to five day-old) and adult(two month-old) normal mice were injected with isogenic myoblasts (onthe right side of the bladder with the same amount of viral particles aswas used to infect myoblasts that were injected into left side ofbladder) and were sacrificed at three days post-injection. Approximately8 animals were assessed in each group. The injected muscles were assayedfor both β-galactosidase activity and for LacZ staining and theefficiency of gene delivery was monitored. After 2, 4, 7, 14, 30, 60,and 90 days, animals were euthanized and the portion of the bladdercontaining the engrafted myoblasts, as well as adjacent normal bladder,were removed and analyzed.

The level of transgene expression was statistically compared between thedirect and the ex vivo gene transfer approaches using adenovirus. Thebladders injected with isogenic myoblasts were longitudinally sectionedto determine the presence of regenerating myofibers. Immunohistologystaining for lacZ and fluorescent microscopy analysis were performed toevaluate and quantify the survival and differentiation of myoblasts andto assess the production of myofibers. The location of transducedmyofibers containing a non-uniform distribution of FLMs allowed thedetermination of the presence of mosaic myofibers. Using immunohistologystaining for LacZ and FLMs analysis, it was determined that there weresurvival and differentiation of injected myoblasts into myofibers.

The results from these analyses demonstrate that autologous myoblastcells can be characterized, cultivated and stored. In addition,autologous myoblasts were shown to be successfully engrafted into thebladder and urethra without significant immunoreactivity for at least 90days up to 6 months. More specifically, the injection of primarymuscle-derived stems cells into the bladder was able to achievelong-term survival and over 50% persistence at 6 months. In addition,maintenance of persistent bulking effect was demonstrated in theurethral wall. Such maintenance is necessary for the treatment of stressurinary incontinence (FIGS. 15A-15C).

Example 5

Further experiments as described in this example were carried out toevaluate and improve the survival of injected myoblasts post-injection.In order to perform this evaluation, different mdx myoblast cell lines(see below), as well as primary myoblasts, at different purities asdescribed below, injected into the muscle of adult mdx mice. Themyoblasts were either adenovirally transduced or transfected with aβ-galactosidase expressing plasmid and the early fate of the injectedcells was evaluated post-injection.

The plasmid containing the gene encoding β-galactosidase was constructedon a pBluescript plasmid backbone, in which the β-galactosidase reportergene is driven by the human cytomegalovirus (HCMV) promoter, and theneomycin-resistance gene is driven by the PGL promoter. (J. A. Wolff etal., 1990, Science, 247:1465-1468; S. Jiao et al., 1992, Human GeneTherapy, 3:21-33).

Different populations of primary myoblasts which had been purified bypreplating (Example 1), and having a percentage of myoblasts rangingfrom between about 10% to 90%, were assayed. Finally, primary mdxmyoblasts obtained from isolated myofibers were also used and comparedamong the groups for the survival of the injected cells (see J. Huard etal., 1994, Human Gene Ther., 5:949-958 and J. Huard et al., 1994, J.Clin. Invest., 93:586-599).

The different populations of preplated myoblast cells were infected withβ-galactosidase expressing adenovirus. The adenovirus, an E1-E3 deletedrecombinant adenovirus kindly obtained through GeneVec Inc. (Dr. I.Kovesdi), had the β-galactosidase reporter gene under the control of thehuman cytomegalovirus (HCMV) promoter and followed by the SV40 t-intronand polyadenylation (Poly A) signal. The adenovirally transduced cellswere then transplanted into the hindlimb muscle (gastrocnemius andsoleus) of mdx mice and assayed for their survival post-implantation.

Myoblasts were isolated from single viable myofibers. Single musclefibers were prepared from dissected extensor digitorum longus (EDL)muscle by enzymatic desegregation in 0.2% type 1 collagenase (Sigma, St.Louis, Mo., USA), as previously described (D. J. Rosenblatt et al.,1995, In Vitro Dev. Biol. 31:773-779; W. G. Feero et al., 1997, GeneTher., 4:371-380). Isolated muscle fibers from 6 week old mice wereused. Following the isolation of approximately 200 myofibers per muscle,a minimum of 5-10 myofibers per well were plated on 6 well plates coatedwith 1 mg/ml Matrigel (Collaborative Biomedical Products, Bedford,Mass., USA) in 2 ml Dulbecco's Modified Eagle Medium (DMEM) containing10% horse serum, 1% chick embryo extract, 2% L-glutamine, and 1%penicillin/streptomycin (Gibco, BRL, Grand Island, N.Y., USA). Theplates were placed in a 37° C., 5% CO₂ incubator for several days. Thecells emerging from these myofibers were grown until confluence, assayedfor desmin expression, transduced with adenovirus carrying LacZ reportergene expression, and tested for the myoblast survival post-implantationfollowing the protocol described below.

An immortalized mdx cell line isolated from a transgenic animal carryinga thermolabile SV40 T antigen under the control of an inducible promoterwas used (J. E. Morgan et al., 1994, Develop. Biology 162:486-498). Theimmortalized mdx cell line proliferated indefinitely under thepermissive conditions of 33° C. with gamma interferon (proliferationmedium; DMEM+20% fetal bovine serum) and underwent normaldifferentiation at 37°-39° C. without gamma interferon (fusion medium;DMEM+2% fetal bovine serum). This myoblast cell line was assayed fordesmin immunoreactivity and the ability to differentiate when cultivatedin a fusion medium. Subsequently, these cells were transduced withadenovirus carrying LacZ reporter gene expression, and the survival ofthe injected myoblasts was analyzed as described below.

Myoblasts were engineered to express anti-inflammatory agents. The mdxmyoblast cell line was used for the genetic engineering of myoblastsexpressing anti-inflammatory substances. The myoblasts were infectedwith a retroviral vector carrying the gene encoding interleukin-1receptor antagonist protein (IL-1Ra) and a neomycin-resistance gene (G.Bandara et al., 1993, Proc. Natl. Acad. Sci. USA, 90:10764-10768).Following infection, the myoblasts were selected using neomycin (1000μg/ml of medium) to obtain nearly 100% infected cells, becausenon-infected cells die when subjected to neomycin treatment.

The selected myoblasts were first analyzed in vitro for their ability toexpress IL-1Ra (80 ng/1×10⁶ cells at 48 hours post-infection). Theengineered myoblasts were found to be capable of differentiating intomyotubes in vitro and of forming myofibers following intramusculartransplantation in vivo. The modified myoblasts were subsequentlyinfected with adenovirus carrying the LacZ reporter gene and injectedinto mdx muscle. The early fate of the injected cells was monitored andcompared with that of non-engineered cells using the protocol describedbelow.

Immunohistochemistry for desmin: The different myoblast populations werefixed with methanol at −20° C. for 1 minute, followed by 2 rinses inphosphate buffer saline (PBS). The cell cultures were blocked with 10%horse serum for 1 hour and incubated with the first antibody (1/200monoclonal mouse anti-desmin, Sigma Co., St. Louis, Mo., USA) for 1hour. Following 3 rinses in PBS, the sections were incubated with asecond antibody (anti-mouse conjugated to Cy3, immunofluorescence,1/200, Sigma Co., St. Louis, Mo., USA). The immunostaining was thenvisualized by fluorescent microscopy and the number of desmin positivecells was computed and compared among the different groups.

Different populations of myoblasts were used for these experiments. 0.5to 1×10⁶ cells were injected percutaneously into the mid-portion of thegastrocnemius muscle for each experimental group; the experimentalgroups that were compared together received the exact same number ofcells. Primary mdx myoblasts, myoblasts isolated from single musclefibers, and immortalized mdx myoblast cultures were transduced with anadenovirus carrying the LacZ reporter gene using a multiplicity ofinfection of 50 (MOI=50). Forty-eight hours post-transduction, thedifferent groups of transduced myoblasts were harvested bytrypsinization (0.1% trypsin), washed in Hank's Balanced Salt Solution(HBSS), and intramuscularly injected with a Drummond syringe. Atdifferent time points post-injection (0.5, 12, 24, 48 hours, and 5days), the animals were sacrificed and the injected muscles were assayedfor LacZ expression (histochemistry and ONPG). The β-galactosidaseexpression obtained from the injected muscles was compared with that ofthe transduced cell extract before transplantation (0 hourspost-injection). Five two-month-old C57BL10/J mdx/mdx mice were used foreach group. The experiment animals were kept in the Rangos ResearchCenter Animal Facility of Children's Hospital of Pittsburgh. Thepolicies and procedures of the animal laboratory were in accordance withthose detailed in the guide for the “Care and Use of Laboratory Animals”published by the USA Department of Health and Human Services.

LacZ staining by histochemical technique: Cryostat sections of theinjected and control muscles were stained for LacZ expression using thetechnique described as follows: sections were fixed with 1.5%glutaraldehyde (Sigma Co., St. Louis, Mo., USA) for one minute andrinsed twice in phosphate-buffered saline (PBS) and incubated in X-galsubstrate [0.4 mg/ml 5-bromo-chloro-3-indolyl-β-D-galactoside(Boehringer-Mannheim, Indianapolis, Ind., USA), 1 mM MgCl₂, 5 mMK₄Fe(CN)₆/5 mm K₃Fe(CN)₆ in PBS] overnight (37° C.). Following the LacZhistochemistry, the muscle sections were counterstained withhematoxylin/eosin and visualized by light microscopy (Nikon, Optiphot.microscope).

β-galactosidase activity was assayed in order to achieve a betterquantification and comparison of the transgene expression level in theinfected cells and the injected muscles (J. Sambrook et al., 1989,Molecular Cloning. A Laboratory Manual, 1.21-1.52.). The injectedmuscles or cells were frozen in liquid nitrogen and homogenized in 0.25M Tris-HCL (pH 7.8), and the homogenates were centrifuged at 3,500×g for5 minutes. The muscle homogenate was disrupted by 3 cycles offreeze/thaw, and the supernatant was centrifuged (12,000×g/5 min. at 4°C.) and transferred to a microcentrifuge tube. 30 μl of this extract wasmixed with 66 μl of 4 mg/ml ONPG (O-nitrophenyl-β-D-galactopyranoside,Sigma Co.) dissolved in 0.1 M sodium phosphate (pH 7.5), 3 μl of 4.5 Mβ-mercaptoethanol dissolved in 0.1 M MgCl₂, and 201 μl 0.1 M sodiumphosphate. The mixture was then incubated at 37° C. for 30 minutes. Thereaction was stopped by adding 500 μl of 1 M Na₂CO₃ and the opticaldensity was read on a spectrophotometer at a wavelength of 420 nm. Thelevel of β-galactosidase activity (# of units/sample) was extrapolatedon a calibration curve, which converted the optical density at 420 nm tothe concentration of β-galactosidase enzyme. The level ofβ-galactosidase enzyme was compared in the transduced non-injectedmyoblasts with that obtained in the injected muscle at 0.5, 12, 24, and48 hours and at 5 days post-injection.

Immunochemical staining for myosin heavy chain (MyHC) isoforms: Onemonoclonal antibody specific for slow myosin heavy chain isoforms wasused. The anti-slow myosin heavy chain (M 8421, Sigma Co., St. Louis,Mo., USA) monoclonal reacts with the slow MyHC of adult skeletal muscle.MyHC staining was performed using indirect immunoperoxidase techniques.10 μm serial cryostat sections were collected on glass slides, fixedwith cold acetone (−20° C.) for 1 minute and blocked with 5% horse serumfor 1 hour. The sections were incubated overnight at room temperature ina humid chamber with primary antibodies diluted 1:500 in PBS, pH 7.4,containing 4% horse serum. The muscle cryostat sections weresubsequently rinsed 3 times in PBS and incubated with sheep anti-mouseantibodies conjugated with horseradish peroxidase (A7282, Sigma Co., St.Louis, Mo., USA), diluted 1:100 in PBS for 90 minutes. After threerinses in PBS, the peroxidase activity was then determined by incubationwith 1 mg/ml diamino-benzidine in PBS containing 0.01% hydrogenperoxide. The peroxidase reaction was then carried out and was stoppedby repeated rinses in PBS. Sections were mounted in GelMount (Biomeda,Corp. Foster City, Calif.) and observed under light microscopy (NikonOptiphot microscope). The co-localization of the LacZ and slow myosinheavy chain-expressing muscle fibers was then performed on serial musclesections.

Statistical analysis: The average transduction level was computed atdifferent time points for each group (n=5) and compared over time byone-factor analysis of variance (ANOVA; multi-comparison type factorial)using statistical software (Stat View 512⁺; Brain power, Calabasas,Calif., USA).

In this example, it was demonstrated that different populations ofprimary muscle-derived cells isolated from gastrocnemius muscle fromdifferent preplates contain a different percentage of desmin positivecells. The different populations of cells consisted of a mixture ofmuscle-derived cells including myoblasts, fibroblasts, and adipocytes.The populations of muscle-derived cells were found to display differentdesmin immunoreactivities, ranging from between 7 to 80% afterpreplating. For example, the first preplate contained 7% desmin positivecells, while the sequential preplates were enriched in their content ofdesmin positive cells (i.e., PP#2=14%, PP#3=25%, PP#4=72%, PP#5=77%, andPP#6=80%).

These cell populations had variable ability to differentiate intomyotubes when cultivated into a fusion medium. In fact, the number ofmyotubes obtained in preplate #1 and preplate #3 was very low comparedwith those obtained in preplate #5 and preplate #6. The resultsdemonstrate that populations of muscle-derived cells with higher numbersof desmin positive cells display a better ability to differentiate intomyotubes.

97% of desmin positive cells were obtained from a single myofiberisolated from Extensor Digitorum Longus muscle (EDL). In addition, themdx myoblast cell line, isolated from transgenic mice carrying the SV40T antigen, was nearly 100% desmin positive. These cells were alsocapable of differentiating into myotubes, which demonstrates the highmyogenicity index of those cell cultures.

Different populations of muscle derived cells isolated and purified fromnormal and mdx (dystrophic) mice by the preplate technique were testedfor the presence of various markers. It was found that a population ofmuscle-derived cells had the following characteristics: about 95% desminpositive; about 95% BCL-2 positive; about 95% CD34 positive; about 95%myosin heavy chain isoforms (MyHCs) expression; about 30-60% MyoDexpression; about 30-60% Myogenin expression; and less than about 10%M-cadherin expression, thereby suggesting their stem cell qualities.

Muscle-derived cells obtained from preplate #1 and injected intogastrocnemius muscle were rapidly lost by 48 hours post-injection: only17% of the expression of the introduced gene present in the injectedmyoblasts was measured in the injected muscle. However, an improvementin the survival of the injected myoblasts was obtained with the use ofcells from subsequent preplates for injection. In fact, the cellsisolated from preplate #2 yielded a 55% myoblast loss at 48 hourspost-injection, those from preplate #3 yielded a 12% myoblast loss, andthose from preplate #6 yielded a 124% gain compared with the level ofLacZ transgene expression present in the cells before injection andtransplantation. (FIGS. 7A-7H). These results suggest that populationsof muscle-derived cells were isolated during preplating that displayed abetter survival rate following transplantation.

Surprisingly, the pure population of myoblasts obtained from theisolated myofibers (fiber myoblasts, Fmb) displaying over 95% desminimmunoreactivity suffered a rapid loss following myoblasttransplantation. In fact, a loss of 96% of the injected myoblasts wasobserved at 48 hours post-transplantation. Similarly, the mdx myoblastcell line (Cell line), (FIGS. 7D and 7H) was rapidly lost followingtransplantation: 93% of the level of LacZ transgene expression presentin the cell culture post-implantation disappeared after 2 dayspost-injection. These results show that the high percentage ofdesmin-positive cells present in the muscle-derived cell population inpreplate #6 was but one factor to explain the improvement of cellsurvival post-implantation.

Even though populations of muscle-derived cells have been isolateddisplaying a better survival post-injection (PP#3, PP#6), all of thecell populations lead to a decrease in reporter gene expression betweendays 2 and 5 post-injection. The cells with the better survival rate,however, retained better transgene expression at day 5.

All of the myoblast populations following adenoviral transduction havebeen found to be capable of delivering the β-galactosidase reporter geneto skeletal muscle at 2 and 5 days post-infection. Using the samenumbers of cells, it was observed that PP#6 and, to a lesser extent,PP#3 offered a better gene transfer than the population ofmuscle-derived cells isolated at preplates #1 and #2. The ability of thepurified muscle cells to circumvent the poor survival of the injectedcells may explain the better efficiency of gene transfer in the injectedmuscle. However, it was also observed that PP#6 displayed a betterability to fuse with host myofibers compared with muscle-derived cellsisolated at earlier preplates. The myoblast cell line (Cell Line) andthe highly pure myoblast culture isolated from myofibers (FMb) alsodisplayed a reduction in gene transfer when compared with themuscle-derived cells isolated at preplate #6, suggesting that theability of cells to bypass poor survival post-injection leads to animprovement of gene transfer to skeletal muscle.

Following injection of muscle cells, the cells either fused together toform myotubes or fused with host myoblasts and muscle fibers to formmosaic myofibers. Serial cryostat sections showed that transducedmyoblasts obtained from isolated myofibers either fused together to formmyotubes and immature myofibers expressing fast myosin heavy chains orfused with host myofibers expressing fast myosin heavy chains. Thissuggested that the myofibers used to isolate myoblasts were probablyexpressing fast myosin heavy chains. In contrast, the muscle-derivedcells isolated at PP#6 fused together and with host myofibers expressingboth fast and slow myosin heavy chains, suggesting that themuscle-derived cells at PP#6 have the ability to fuse with fast and slowmyosin heavy chains myofibers.

To investigate whether the cells capable of expressing anti-inflammatorysubstances can improve the cell survival post-injection, myoblasts weregenetically engineered to express interleukin-1 receptor antagonistprotein (IL-1 Ra), which is able to compete with inflammatory cytokineIL-1 for binding to the IL-1 receptor, but does not induce IL-1 receptorsignalling. The survival of the engineered myoblasts was then comparedwith that of non-engineered control cells. The myoblast used for thisexperiment, the mdx cell line, displayed a drastic loss of the injectedcells post-injection (FIG. 7G). An 84% loss of the non-engineered cellswas observed by 48 hours post-injection through the significant decreasein the amount of β-galactosidase expression in comparison to thenon-injected transduced myoblast at time 0. Moreover, a slight increasein the amount of reporter gene was detected at 120 hours post-injection,which remains significantly different from that observed in controlcells (FIG. 8A).

In contrast, the cells engineered to express IL-1Ra, significantlyreduced the early loss of the injected cells. A loss of 60% in theamount of β-galactosidase expressed in the non-injected cells (P<0.05)at 24 hours post-injection was seen, but, in contrast to the resultobserved with the non-engineered cells, the level of β-galactosidaseexpression detected at 2 days post-injection was not found to besignificantly different than that found in the non-injectedIL-1Ra-expressing myoblasts. This finding suggests that inflammationalso plays a role in the poor survival rate of the injected cells;consequently, approaches capable of blocking the inflammation may aid inthe development of strategies to achieve efficient myoblasttransplantation (FIGS. 8B-8D).

The experiments in this example were performed to determine whetherinflammation is the only factor involved in the poor survival of theinjected myoblasts. The extremely wide range of biological activities ofIL-1Ra may improve the cell survival by blocking the action of aninflammatory cytokine (IL-1). As described herein, myoblasts weregenetically engineered to express anti-inflammatory IL-1Ra, and weretested for the prevention of rapid loss of the injected cells. Theengineered myoblasts expressing IL-1Ra allowed for a better survivalrate of the injected myoblasts at 48 hours post-injection. Thenon-engineered myoblast cell line displayed poor survival of theinjected cells, but the same cell line expressing IL-1Ra significantlyimproved the survival rate of the injected cells.

An 80% improvement in the survival of the injected cells was observed bythe local expression of an anti-inflammatory substance. There was asignificant decrease in the amount of the β-galactosidase reporter geneat 24 hours compared with the non-injected control cells, suggestingthat IL-1Ra expressing myoblasts probably fused between 24 and 48 hourspost-injection and led to an increase of the expression ofβ-galactosidase reporter gene. A slight decrease in the amount ofβ-galactosidase reporter gene expression was observed at 5 dayspost-injection which may be related to immune rejection.

A substantial reduction in the loss of the injected myoblasts wasachieved with the IL-1Ra-expressing cells, but a 20% loss was stillobserved at 48 hours post-injection. Thus, inflammation is likely to beonly one factor involved in the rather rapid loss of injected myoblastspost-implantation.

Different populations of muscle-derived cells were evaluated to assistin the development of approaches to circumvent the poor survival ofinjected myoblasts post-transplantation. To carry out this evaluation,mdx myoblast cell lines, pure myoblasts isolated from myofibers, andprimary muscle-derived cells at different purities were injected intoadult mdx muscle. The myoblasts were adenovirally transduced asdescribed, and the early fate of the injected cells post-injection wasevaluated at different time points post-injection (i.e., 0.5, 12, 24, 48hours, and 5 days). It was observed that populations of primarymuscle-derived cell cultures isolated from a gastrocnemius muscledisplayed a differential ability to express desmin and differentiateinto myotubes. In addition, the cell survival post-injection wasdifferent. In fact, the same number of muscle cells derived frompreplate #1 versus preplate #6 resulted in an 83% loss and a 124% gain,respectively, in the transgene expression, compared with non-injectedtransduced cells at time 0. This suggests that the isolation of specificpopulations of muscle-derived cells totally overcomes the rapid lost ofthe injected cells.

This example evaluated whether the source of muscle-derived cells couldhave a primordial role in the early survival of the injected myoblasts.A great difference in the content of satellite cells has already beenobserved between slow and fast twitch muscles (H. Schmalbruch and U.Hellhammer, 1977, Anat. Rec., 189:169; A. M. Kelly, 1978, Dev. Biol.,65:1; M. C. Gibson and E. Schultz, 1982, Anat. Rec., 202:329). The typesof satellite cells isolated from these muscles may possibly display adifferential fate post-transplantation.

The PP#6 muscle-derived cells were found to have the ability to fusewith myofibers expressing both slow and fast myosin isoforms, incontrast to myoblasts obtained from isolated myofibers, which eitherfused together or fused with myofibers expressing the fast myosinisoform. The inability of myoblasts obtained from isolated myofibers tofuse with myofibers expressing the slow myosin isoform may be involvedin the differential survival of the injected myoblasts, since theinjected muscle (gastrocnemius) contains a mixture of myofibersexpressing fast and slow myosin isoforms. The injected myoblasts thatare unable to fuse with host myofibers will likely die at the injectionsite. Without wishing to be bound by theory, this may explain why themyoblast cell line displaying a high desmin immunoreactivity still showsa poor survival post-injection.

Even though different populations of muscle-derived cells and the IL-1Raexpressing myoblasts display a better survival post-implantation inskeletal muscle, the long-term persistence of the injected cells appearsto be hindered by immune responses. In general, all populations ofmuscle cells display a reduction in the amount of β-galactosidaseexpression between day 2 and 5 post-injection. It was also found thatthe number of LacZ-expressing myofibers decreased over time in theinjected muscle, even when IL-1Ra expressing myoblasts were used.Consequently, the engineering of specific populations of muscle-derivedcells not only to express IL-1Ra, but also to express additional immunesuppression factors or agents could likely prevent both poor survivaland any adverse immune responses that may accompany myoblast transfer.

Example 6

Experiments were performed to determine the feasibility of cell mediatedgene therapy using myoblasts transduced by an adenovirus vector carryingthe gene encoding basic fibroblastic growth factor (bFGF). bFGFengineered myoblasts were injected into the urethra to treat stressincontinence and also into the bladder wall to improve detrusorcontractility. Adenoviral vectors used were produced as described by H.Ueno et al., 1997, Arteriosclerosis, Thrombosis & Vascular Biology,17(11):2453-60 and J. C. Takahashi et al., 1997, Atherosclerosis,132(2): 199-205.

Experiments similar to those described for urethra and bladder injectionin Examples 2 and 3 were performed to compare sham control animalsversus animals injected with myoblasts transduced with adenovirusvectors genetically engineered to contain the bFGF gene; the transducedmyoblasts expressed bFGF protein following transduction.

Briefly as described, four groups of animals were assessed: controlbladder and control urethra animals with myoblast-bFGF or sham salineinjection. Approximately 8 animals were evaluated in each group. Theanimals were sacrificed 4 days after injection. Animals which hadreceived urethral injection underwent physiological assessment asdescribed in Example 2. Animals which had received bladder injectionunderwent physiological assessment as described in Example 3.

In these experiments, myoblasts were transduced with an adenovirusvector carrying both the bFGF gene and a β-galactosidase reporter gene,described above, and were also labeled with fluorescent latexmicrospheres (FLMs) (P. Cuevas et al., 1996, Proc. Natl. Acad. Sci. USA,93:11196-12001; Y. Nakahara et al., 1996, Cell Transpl., 5:191-204).Histochemical staining for bFGF, lacZ and fluorescent microscopicanalysis was performed to evaluate and quantify the survival anddifferentiation of myoblasts and to assess the generation of myofibers.

Example 7

The experiments described in this Example demonstrate that myoblastsinfected with an adenovirus vector carrying the iNOS gene and injectedlocally into the urethra and bladder decrease bladder inflammation,improve urethral relaxation, and affect erectile dysfunction.

Briefly, and in accordance with the present invention, myoblasts wereinfected with adenovirus engineered to carry the iNOS gene (Example 1).The transduced myoblasts were injected into either the urethra orbladder of S.D. rats. Physiological experiments as described in Examples2 and 3 for the bladder and urethra were performed to assess thephysiological effect(s) of iNOS gene therapy in these tissues.

The cyclophosphamide (CYP) model was employed for bladder inflammationand a partial urethral ligation model was employed for partial urethralobstruction. For the CYP bladder inflammation model, 4 groups of animalswere assessed: control and CYP animals received myoblast-iNOS or shamsaline bladder injections. Approximately 8 animals comprised each group.CYP injection (100 mg/kg i.p.) was performed at the same time asmyoblast-iNOS or sham injection. The animals were sacrificed 4 daysafter the injections, based on determinations of persistent bladderinflammation beyond 4 days after CYP, and optimal iNOS expression at 4days (E. Tzeng et al., 1995, Proc. Natl. Acad. Sci. USA, 92:11771-11775;E. Tzeng et al., 1996, I. Surgery, 120:315-321).

For the urethral obstruction model, 4 groups of animals were alsostudied: control and urethral ligation animals with myoblast-iNOS orsham saline urethral injection. Myoblasts were transduced with anadenovirus vector carrying the β-galactosidase reporter gene and werelabeled with fluorescent latex microspheres (FLMs). Histochemicalstaining for lacZ and fluorescent microscopic analysis were performed toevaluate and quantify the survival and differentiation of myoblasts andto assess myofiber development. Immunohistochemistry staining for iNOSand for adenovirus proteins was performed in all specimens. Northernblot analysis for human iNOS RNA production was assessed ingene-transfected animals and in the appropriate controls.

Experiments and the results thereof related to intraurethral andintravesicle NO production are presented more specifically below:

A. Intraurethral NO Donors Relax Urethral Smooth Muscle

To first assess the role of NO donors on smooth muscle, urethralperfusion pressure and isovolumetric bladder pressure were measured withcatheters inserted through the bladder dome in urethane anesthetizedfemale S.D. rats (250-300 grams), (n=9). The catheter assembly waswedged in the bladder neck to block passage of fluid between the bladderand urethra without affecting the nerve supply to the organs. Theexternal urethra was not catheterized. Responses were examined in thecontrol state at an urethral saline infusion speed of 0.075 ml/min.After blocking striated urethral sphincter with intravenousalpha-bungarotoxin administration (100 mcg/kg), intraurethral drugs wereadministered.

The results of these experiments revealed that the urethra exhibitedreflex responses characterized by an initial decrease in urethralpressure at the onset of reflex bladder contractions. This was followedby a period of high frequency oscillations which was abolished byalpha-bungarotoxin. Intraurethral infusion of NO donors, (i.e.,S-nitroso-N-acetylpenicillamine [SNAP] (2 mM) and nitroprusside (1 mM)),immediately decreased baseline urethral pressure by 30% to 37% andmaximum urethral relaxation by 24% to 50%. In addition, the duration ofreflex urethral relaxation was increased by 96% to 100%, which waslikely to be due to exogenous NO potentiating endogenous NO relaxation.Neither NO donor changed the amplitude of bladder contraction. Theurethral relaxation persisted for 15 minutes after stopping the infusionof NO donors. Intraurethral NO donors did not affect the mean bloodpressure. Intraurethral N-nitro-L-arginine (L-NAME) (20 mM) onlypartially blocked the endogenous NO reflex relaxation.

The results of these experiments demonstrate that NO plays a key role inreflex urethral smooth muscle relaxation during micturition. Topicalintraurethral NO donors rapidly induced urethral relaxation withoutaffecting the bladder. On the basis of these results, intraurethralapplication of NO donors can be clinically effective in cases ofurethral smooth muscle sphincter spasticity and obstruction.

B. Intravesical NO on Cyclophosphamide-Induced Cystitis

To first assess the role of intravesicle NO on cyclophosphamide-inducedcystitis, female S.D. rats received a single intraperitoneal injectionof CYP (100 mg/kg) or vehicle. Voiding parameters (mean voided volumeand number of voids) were then monitored for 24 hours in a metaboliccage. Forty-eight hours after CYP injection, bladder function wasstudied using continuous saline infusion (0.04 ml/min) cystometry viaPE-50 transurethral catheter under urethane anesthesia. The NO donorsS-Nitroso-N-acetyl-penicillamine (SNAP) (2 mM) and sodium-nitroprusside(SNP) (1 mM), as well as the NOS inhibitor N-nitro-L-arginine methylester (L-NAME) (20 mM), were administered intravesically.

The results showed that the number of micturitions during the 24 hoursafter CYP injection was significantly greater for 24 hours (117±86, n=8)than for control rats (25±4, n=4) (p<0.001). After 7 days, the voidingfrequency was still persistently elevated (38±4, n=4) (p<0.05). Therewas no difference in total micturition volume between CYP treated(20.5±9.2 ml) and control rats (15.2±6.1 ml). Infusion of an NO donor(SNAP: n=5 or SNP: n=3) increased the intercontraction interval (ICI)(64%), but did not change the amplitude of bladder contractions.Treatment of L-NAME (n=8) did not alter ICI or amplitude (Table 3).

In addition, the CYP injected rats were monitored for over 7 days anddemonstrated persistently elevated voiding frequency, consistent withbladder irritation.

TABLE 3 Contraction duration ICI (min) Amplitude (cmH₂O) (min) Saline4.4 ± 2.3 23 ± 8 1.1 ± 0.3 L-NAME 3.8 ± 2.0 21 ± 6 1.0 ± 0.4 NO donor 7.2 ± 4.6* 21 ± 5 1.2 ± 0.3 *Infusion of NO versus saline and L-NAME: p< 0.01 (Paired t-test)

The foregoing experiments demonstrated that CYP caused long-term bladderirritation and that intravesical NO donors acutely and effectivelyreversed the CYP-induced bladder activity. Without wishing to be boundby theory, it is hypothesized that this drug effect is not due to asmooth muscle relaxation, but rather is mediated by a change in afferentnerve excitability which increases the intercontraction interval, butdoes not change contraction amplitude. Topical NO donors may beconsidered as a treatment for cyclophosphamide and other inflammatorymediated cystitis.

C. Myoblast mediated iNOS gene therapy in the (genito)urinary system

Experiments were then performed to demonstrate that iNOS gene therapyaccording to the present invention is advantageous and efficacious fortherapeutic use in the genitourinary system.

1. iNOS Gene Therapy for Erectile Dysfunction: Comparison Among Plasmid,Adenovirus and Adenovirus Transduced Myoblast Vectors

The gene encoding the inducible form of NOS (iNOS) was inserted into aplasmid and into an adenovirus vector. The iNOS plasmid was as describedin D. K. Nakayama et al., 1992, Am. J. Respir. Cell. Mol. Biol.,7:471-476. The adenovirus iNOS vector was as described in D. A. Gelleret al., 1993, Proc. Natl. Acad Sci USA, 90:3491-3495 and in U.S. Pat.Nos. 5,658,565 and 5,468,630. By injecting a solution of either plasmid,virus, or virally transduced myoblasts, exogenous human iNOS wasintroduced into the rat penis.

Adult male rat penis was injected with myoblasts transfected withadenovirus vectors carrying the β-galactosidase gene. After 2, 4 and 7days, X-gal staining and the physiological effects of X-gal wereassessed. 100 μl injections of adenovirus with titers of 10⁶-10⁹ weretested. Myoblasts were plated at a density of 5×10⁴ cells per well andwere transduced with adenovirus stock (MOI=50). Injections ranged from1.33×10⁵ to 1×10⁶ cells per 100 μl. Basal intracavernous pressure (ICP)and maximal ICP after cavernous nerve stimulation were measured. iNOSimmunohistochemistry and PCR of human iNOS primer were also performed.For plasmid experiments, 100 μg of plasmid injected in 100 μl of 20%sucrose/PBS was used for injection into the bladder, penis and urethra.

Optimal conditions for gene transfection were determined by varying thenumber of virus particles (pfu), concentration, volume and vehicle ofthe injected solution. The level of iNOS or reporter gene expression wastime dependent. Maximal expression was found at day 4, with lower levelsat day 2 and minimal levels at day 7. At day 4, gene expression wasgreatest for myoblast+adenovirus>adenovirus>plasmid. Immunohistochemicalstaining of iNOS and adenovirus proteins was detected in treated penis.Northern blot analysis for human iNOS was positive only ingene-transfected animals. There was no difference in basal or maximalICP between control and animals treated with the β-galactosidasereporter gene. However, there was a significant increase in basal ICP(55±23 cmH₂O) in gene treated penis versus control (5±6 cmH₂O). MaximalICP increased two fold in iNOS transfected rats.

The results demonstrated that myoblast mediated gene therapy was moresuccessful in delivering iNOS into the penis than was direct virus orplasmid transfection methods. NOS gene therapy according to the presentinvention thus promises to provide a new treatment for erectiledysfunction.

2. Measurement of iNOS Gene Therapy

The release of NO was confirmed using a porphyrinic microsensor placeddirectly in tissues treated with inducible NOS (iNOS) gene therapy (L.A. Birder et al., 1998, American Urological Association Meeting, 1998).Ex vivo gene transfer was used to place the human iNOS gene into thepenis and bladder of male S.D. rats. Injections of 100 μl of infectedmyoblasts suspended in saline solution at a total cell count of 1.33×10⁵to 1×10⁶ cells per injection were injected into the corpora cavernosumof the rat. The bladder was injected with a similar solution; however,the cell concentration ranged from 1.33×10⁵ to 1×10⁶ cells per 10 μlinjection. Transduced myoblasts were prepared by incubating them for 24hours with adenovirus vector containing the iNOS gene at a multiplicityof infection of 50 virus particles per cell. Immunohistochemicalstaining was performed on the cultured cells to ensure adequateinfection and iNOS protein production.

NO release in the bladder and penis was measured by placing the tip of aNaflon-coated porphyrinic microsensor (dia. 10 μM; detection limit, 5nM; response time, 1 ms) directly on the bladder surface (mucosal andserosal) and on the corporal cavernosum, respectively. The NO releasepeak concentration of 1-1.3 μM was evoked by the adrenergic agonist,norepinephrine (3 μM) and also by the iNOS cofactor, tetrahydrobiopterin(TBH4).

Incorporation of the iNOS gene into the penis and bladder was detectedby positive immunohistochemical staining for iNOS and adenovirusantibodies, and Northern blot analysis for human iNOS RNA production waspositive only in human iNOS gene transfected animals. The porphyrinicmicrosensor measured low but similar levels of constitutive NO releasein various areas of the bladder. The addition of 100 μM TBH4 caused abrief (<5 second) spike in NO release (1-5 nM) in control areas of thebladder.

In iNOS gene injected areas, there was a sustained release (>1 min.) oflarge amounts of NO (>20 nM). The porphyrinic microsensor demonstratedincreased NO release in areas of the bladder treated with myoblast cellmediated iNOS gene therapy.

Example 8

The use of co-factors such as trophic factors, i.e., cytokines,expressed by myoblasts genetically engineered to contain genes encodingsuch factors is a beneficial aspect of the present invention.

Muscle-related injuries are a challenging problem in many fields ofmedicine. Even though muscles retain their ability to regeneratefollowing injury, the healing process of muscles following such injurieshas been found to be very slow and often leads to an incomplete musclerecovery. To develop approaches to improve muscle healing followinginjury, e.g., bladder or urethral wall injury (cryoinjury), the presentinventors have developed reproducible injury models for musclecontusion, strain, and laceration.

Investigations related to the present invention have shown that muscleregeneration occurs following the above-mentioned injuries, but that thedevelopment of scar tissue formation greatly limits the natural healingprocess. Thus, it was newly determined that an enhancement of musclegrowth and regeneration could be used to improve muscle healingfollowing injuries. Accordingly, growth factors were identified whichenhanced not only myoblast proliferation and differentiation in vitro,but also muscle regeneration in injured muscles which improved musclehealing following injuries. These findings can also have directapplication in cases of lower urinary tract smooth muscle injuryassociated with stress urinary incontinence and impaired bladdercontractility.

A. Development of Animal Models for Muscle Injury

The characterization of approaches to improve muscle healing followinginjuries required the development of well-defined, reproducibleorthopaedic muscle injury models, including contusions, lacerations, andstrains as described herein. The characterization of muscle regenerationfollowing these injuries allowed the determination of the muscle'snatural healing following injury. The muscle laceration, contusion, andstrain models were developed in mice.

Briefly, the muscle laceration was performed by cutting thegastrocnemius muscle of a mouse with scissors at 60% of its length fromits distal insertion, 75% of its width, and 50% of its thickness. Themuscle contusion was made by dropping a 16 gram ball through an impactorfrom a height of 100 centimeters onto the mouse's gastrocnemius muscle,and the muscle strain was performed by elongating the muscle-tendon unitto a pre-determined strain point at the rate of 1 cm/min.

In order to evaluate muscle healing following injury, histologicalstaining (hematoxylin-eosin) in conjunction with immunohistochemicaltechniques were employed to assess the expression of desmin andvimentin. Since desmin is a cytoskeletal protein uniformly expressed inregenerating myofibers, it has been used to specifically locateregenerating myofibers. Vimentin is expressed in mononucleatedfibrocytes and macrophages and has been used herein as a marker forfibrosis.

It was observed that the injured muscle was capable of healing due to amassive muscle regeneration which occurred following injury. The highlevel of muscle myofiber regeneration seen at 7 and 10 days followinginjury began to decrease after about 14 days and continued to decreaseuntil 35 days post-trauma. The development of a large scar tissueformation in the injured muscle revealed that the muscle healing was notcompleted at 35 days post-injury. In fact, it was observed that thedevelopment of the scar tissue formation started at 14 days post-injuryand increased gradually until 35 days. It is likely that the developmentof fibrosis, which appears to hinder the healing process, can be relatedto the reduction of muscle regeneration which is also observed at 14days post-injury.

B. Characterization of the Effects of Several Growth Factors on MyoblastProliferation and Fusion In Vitro

In approach for improving muscle healing following injury is toaccelerate muscle regeneration. One way to achieve this acceleration isby increasing the myogenic activity of muscle cells in the injuredmuscle. Substances which enhance myoblast proliferation anddifferentiation in vitro may also increase muscle regeneration in vivoand prevent the development of scar tissue formation.

During muscle regeneration, numerous growth factors are released by theinjured muscle to modulate muscle regeneration. These proteins activatethe satellite cells to proliferate and differentiate into myofibers tosupport muscle regeneration (E. Schultz, 1989, Med. Sci. Sports Exer.,21:181; T. Hurme and H. Kalimo, 1992, Med. Sci. Sports Exer.,24:197-205; R. Bischoff, The satellite cell and muscle regeneration.Myology. 2nd Edition. New York, McGraw-Hill, Inc, pp. 97-118, 1994).During growth and development of skeletal muscle, many growth substanceshave been found to be capable of inducing various responses from theskeletal muscle. In fact, the individual effects of these growth factorson specific steps of muscle regeneration have been shown (R. L. Chambersand J. C. McDermott, 1996, Can. J. Appl. Physiol., 21:155-184; J. R.Florini and K. Magri, 1989, Am. J. Physiol., 256:701-711; M. D. Ground,1991, Path. Res. Pract., 187:1-22).

Several growth factors, including acidic and basic fibroblast growthfactors (aFGF and bFGF); epidermal growth factor (EGF); insulin-likegrowth factor-1 (IGF-1); platelet derived growth factor (PDGF);transforming growth factor β or α (TGF-β or TGF-α); and nerve growthfactor (NGF) have been investigated for their ability to enhance themyogenic activity of muscle cells in vitro. Myoblasts were cultured withthese trophic factors at different concentrations (1, 10 and 100 ng/ml),and the myoblast proliferation and differentiation were monitored at 48and 96 hours post-incubation. The findings herein revealed that b-FGF,IGF-1 and NGF each significantly enhanced myoblast proliferation invitro.

Further, bFGF, aFGF, IGF-1, and NGF were found to be able to increasemyoblast differentiation into myotubules (Table 4). Other growth factorswere incapable of significantly increasing either myoblast proliferationor differentiation. These results showed that bFGF, NGF, and IGF-1significantly enhanced myoblast proliferation, while b-FGF, a-FGF IGF-1and NGF increased myoblast differentiation into myotubules. Thus, thesefactors afford beneficial results when delivered into an injured muscleto improve muscle healing following injury in accordance with thepresent invention (Table 5).

TABLE 4 Effect of Growth Factors on Myoblasts in vitro ProliferationFusion b-FGF, IGF, NGF Stimulate Stimulate a-FGF Inhibit Stimulate PDGF,EGF, TGF-α, Inhibit Inhibit TGF-βC. Direct and Ex Vivo Gene Transfer of Selected Trophic Factors

The technique chosen to deliver prospective growth factors to injuredmuscle is of paramount importance to optimize therapeutic benefit.Options include direct injection of growth factors, direct gene therapy,ex vivo gene therapy, and myoblast transplantation.

Ex vivo delivery of the β-galactosidase marker gene to injured muscleproduces many β-galactosidase positive myofibers. The ex vivo musclecell-mediated approach provides not only an efficient method ofdelivering selected genes, but also provides cells capable ofparticipating in the reparative process, similar to myoblasttransplantation. However, myoblast transplantation lacks in vitrogenetic manipulations. In addition to its application toward inheritedmuscle diseases, myoblast transplantation is shown to improve myofiberregeneration in muscle experimentally injured with myonecrotic agents.Therefore, the closely related techniques of muscle cell-mediated exvivo gene therapy and myoblast transplantation are both applicable tomuscle injuries.

Individual direct injections of b-FGF, IGF-1, and NGF into injuredmuscle (e.g., laceration, contusion, and strain) can increase the numberof regenerating myofibers in vivo and increase both muscle twitch andtetanic strength 15 days after injury. However, secondary to rapidclearance and short half-lives, the effect of direct growth factorinjections is likely transient and suboptimal, due to the rapidclearance of the product from the injured sites and the short half lifeof these proteins. By contrast, persistent gene expression through cellmediated gene therapy in accordance with the present invention canfurther improve muscle healing following injuries. The engineering ofvectors capable of expressing these growth factors opens new avenues oftreatment for injured muscles. Also in accordance with the presentinvention, gene therapy based on direct and ex vivo gene transfer iscapable of delivering a gene (e.g., a marker gene) in the injured muscle(i.e., laceration, contusion, or strain).

Direct gene therapy to deliver genes to skeletal muscle is possibleusing naked DNA retrovirus, adenovirus, herpes simplex virus andadeno-associated virus. Most of these vectors transduce relatively fewadult myofibers. However, adenovirus is capable of transducing a largenumber of regenerating muscle fibers, a condition present in injuredmuscle.

Direct injection of adenovirus containing the beta-galactosidase markergene into lacerated, contused, and strained muscle results in manytransduced myofibers at 5 days. Moreover, direct injection of adenoviruscarrying growth factor genes (i.e. bFGF, IGF-1, NGF) should result insustained protein production in injured muscle. Direct injection ofadeno-associated virus (AAV) results in a high level of adult myofibertransduction in both injured and non-injured muscle AAV may be thepreferred vector for direct gene delivery to mature skeletal muscle,although it is capable of carrying genes of only 1-4 KB pairs.

According to the practice of the present invention, the injection ofengineered muscle-derived cells expressing IGF-1 was capable ofimproving muscle healing following laceration injury whenintramuscularly injected at the injured site. The injected muscle withmuscle-derived cells expressing IGF-1 displayed a higher fast twitch andtetanic strength than the injured muscle treated with saline. Theseresults suggest that the injection of muscle-derived cells engineered toexpress IGF-1 can be used to improve muscle healing followingorthopaedic injuries, including laceration, contusion, strain, ischemiaand denervation (Table 5). Since adenovirus has been found immunogenic,an adeno-associated virus was constructed to carry the gene forexpression of IGF-1 (FIG. 12). This virus is used to engineer musclecells that will be injected after engineering in vitro to express IGF-1in the injured muscle and consequently improve muscle healing followinginjury.

It was also determined that myoblast mediated ex vivo gene transfercould efficiently deliver β-galactosidase expressing adenovirus ininjured muscle. In fact, the myoblast mediated ex vivo approach resultedin the production of many β-galactosidase positive myofibers in theinjured muscle following laceration, contusion, and strain. Accordingly,injected myoblasts expressing growth factors are likely to improvemuscle regeneration by the production of growth factors, and to serve asa reservoir of additional myoblasts that are capable of forming newmyofibers.

TABLE 5 Effect of Growth factors on twitch/tetanic strength Effect ofTrophic Factors on Muscle Strength In Vivo NGF bFGF IGF-1 Musclecontusion  +/+* +/+ +/+ Muscle laceration −/− +/+ +/+ Muscle strain +/−+/+ +/+ *(+) improvement in strength vs. non-injected muscles; (−) noimprovement in strength vs. non-injected muscles.D. Myoblast Transplantation to Enhance Muscle Regeneration and ImproveMuscle Healing Following Injury

Myoblast transplantation, which consists of the implantation of myoblastprecursors (satellite cells), enhances muscle regeneration and creates areservoir of normal myoblasts that can fuse and deliver genes toskeletal muscle.

To improve muscle healing following injury, muscle biopsy is obtainedfrom a non-injured muscle of the same individual that will serve as anautologous donor for the myoblast transfer of the injured muscle. Theuse of autologous myoblast transfer can circumvent the well documentedimmune rejection problem, which is a major hurdle of myoblasttransplantation (J. Huard et al., 1994, Human Gene Ther., 5:949-958; J.Huard et al., 1994, J. Clin. Invest., 93:586-599), and lead to improvedmuscle healing following injury.

Example 9

Myoblast-mediated cell transfer for improving, reducing or eliminating anumber of orthopedic conditions was assessed in this example. The animalmodels used were newborn rabbit and adult SCID mice. Such experimentspoint to the utility of myoblast cell-mediated gene therapy forpathologic conditions of the musculoskeletal system, for example,arthritis and damage to joints, ligaments, cartilage and meniscus.

The present inventors have performed extensive numbers of experimentsexploring myoblast mediated gene transfer for orthopedic conditions.Although ex vivo gene transfer using synovial cells has been shown todeliver genes encoding anti-arthritic proteins into the rabbit kneejoint, success using synovial cells has been limited by a transientexpression of the transgene. In accordance with the present invention,muscle cells were employed as an alternative gene delivery vehicle tothe joint in both newborn rabbits and adult SCID mice. It wasdemonstrated that myoblasts were transduced with a higher efficiencythan synovial cells using the same adenoviral preparation toinfect/transduce the cells in vitro. Following intra-articular injectionof genetically engineered muscle cells, the engineered myoblasts adheredto several structures in the joint, including the ligament, capsule, andsynovium. In addition, myoblasts fused to form many post-mitoticmyotubes and myofibers at different locations of the newborn rabbitjoint 5 days post-injection.

In the knees of adult SCID mice, myoblasts fused and expressed thereporter gene for at least 35 days post-injection. The presence ofpost-mitotic myofibers in the knee joint reveals the advantages of thepresent invention for long term expression of secreted protein.Currently, numerous tissues in the joint (ligament, meniscus, cartilage)have poor intrinsic healing capacity and frequently need surgicalcorrections. A stable gene delivery vehicle to the joint, which producesproteins that ameliorate these different musculoskeletal conditions,provides a change for the clinical implications of joint pathologies.

A. Preparation of Primary Myoblasts and Synovial Cells

Muscles from newborn (2-day-old) rabbit legs were removed, and muscletissue was isolated from other connective, vascular, cartilaginous andbony tissues under a dissecting microscope. The isolated myogenic tissuewas dissociated by enzymatic treatment with collagenase 0.2% (1 hour)and trypsin 0.1% (30 minutes) to isolate satellite cells. The primarycultures of myogenic cells were enriched by preplating (1 hour) the cellsuspension in 24-cm² tissue culture flasks coated with 2% gelatin. Sincefibroblasts tend to adhere to the substratum faster than myoblasts, thesupernatant containing myoblasts and other cell types was replated andmaintained in DMEM medium containing 20% fetal bovine serum (FBS) forthree days to obtain approximately 5×10⁶ myoblast cells per flask.

The primary cultures of synovial fibroblasts were prepared fromdissected synovia of adolescent New Zealand White rabbits. The cellswere grown in Ham's F-12 medium supplemented with 10% FBS (S. Floyd etal., 1997, Basic Appl Myol., 7(3&4)).

B. Preparation of Myoblasts and Synovial Cells from Immortalized CellLines

The mdx myoblast cell line was isolated from a transgenic mdx mousecarrying a thermolabile SV40 T-antigen under the control of an induciblepromoter (J. Huard et al., 1994, Human Gene Ther., 5:949-958; J. Huardet al., 1994, J. Clin. Invest., 93:586-599). The permanent mdx cell lineproliferates indefinitely under permissive conditions (33° C. with gammainterferon) and undergoes normal differentiation at 37-39° C. withoutgamma interferon. The immortalized, rabbit-derived synovial cell lineHIG-82 (H. I. Georgescu et al., 1988, In Vitro Cell. Dev. Biol.,24:1015-1022) was propagated in Ham's F-12 medium supplemented with 10%FBS.

C. Comparison of the Transduction Efficiency of the Different Cell Types

Once all of the cell types were prepared (i.e., primary and immortalizedmyoblasts and primary and immortalized synovial cells), 50,000 cells ofeach type were plated in 6 well plates. Each well was then incubated for48 hours with an adenoviral vector carrying the LacZ reporter gene underthe control of the human cytomegalovirus promoter (AV-HCMV-LacZ fromGenvec) at a multiplicity of infection of 25 (MOI=25). The cells werethen stained for the presence of β-galactosidase using X-galhistochemistry. The amount of β-galactosidase activity was alsoquantified using the LacZ assay (J. Huard et al., 1997, Human GeneTher., 4:439-450).

D. Myoblast Differentiation In Vitro and Desmin Staining

Some myoblast cultures were allowed to differentiate by using a fusionmedium containing DMEM supplemented to contain 2% FBS. This fusionmedium reduced cellular proliferation and promoted myoblast fusion.After myotubes had formed in culture, X-gal staining was also performed.In addition, standard desmin (muscle specific marker)immunohistochemistry was also performed to validate the myogenicityindex of these cell cultures (J. Huard et al., 1995, Gene Therapy,2:1-9; D. K. Booth II et al., 1997, J. Tissue Eng., 32:125-132; J.VanDeutekom et al., Human Gene Therapy, 1997).

E. Determination of the Early Fate of the Muscle Cells Injected into theNewborn Rabbit Joint

Primary synovial cells, immortalized synovial cells, primary musclecells and immortalized myoblast cultures were first infected with 25 MOIof AV-HCMV-LacZ for 24 hours as described above. After viral infection,the cells were rinsed and further incubated with a 1:1000 dilution ofFluorescent Latex Microspheres (FLMs), (Microprobes, Inc.), for anadditional 12 hours. The FLMs are fluorescent microspheres which arephagocytized by cells and can serve as an additional marker to followthe early fate of the cells injected into the joint (A. Satoh et al.,1993, J. Histochem. Cytochem., 41:1579-1582).

The infected cells incubated with FLMs were injected into the knees ofnewborn rabbit pups. Each flask of 1×10⁶ cells was trypsinized using0.5% Trypsin-EDTA, centrifuged at 3500 rpm for 5 minutes, andresuspended in 100 μl of Hank's Balanced Salt Solution (HBSS). Thenewborn pups were anesthetized using methophane inhalation for 1 minute.100 μl solutions of infected muscle cells (1×10⁶ cells) were injectedinto the knee joint via the patellar tendon using a 30 gauge needle. Atotal of 10 rabbit pups were used: 2 (4 knees) injected with the primarymuscle cells; 2 (4 knees) injected with the immortalized synovial cells;4 (8 knees) injected with the immortalized myoblasts; 1 (2 knees)injected with saline; and 1 (2 knees) served as a sham, non-injectedcontrol.

The pups were sacrificed five days post-injection, and the knees removedin their entirety. The knees were snap frozen and cryostat sectioned intheir entirety in 10 μm thick slices. Various analyses were performed onthese knee sections, including: histological staining(hematoxylin-eosin); detection of β-galactosidase; localization of FLMs;and desmin immunofluorescence labeling. The distribution of the FLMs anddesmin was visualized using fluorescent microscopy (Nikon Optiphot-2).Co-localization of FLMs and β-galactosidase was used to substantiate thetransgene expression from the injected cells. This additional markerminimized the chances of false positive results which often occur due toendogenous LacZ expression. In addition, myoblast fusion into theintra-articular structures was investigated using desmin labeling.

Newborn rabbits were used for this phase of analysis because their kneesdo not begin calcifying until 15 days of age. Thus, it is possible toflash freeze them in their entirety and analyze the entire kneesystematically. This systematic analysis of cryostat sectionsdemonstrating gene therapy to the joint has not been done previously.This approach permitted the characterization of the differentintra-articular structures to which the transduced cells had adhered.

F. Determination of Long Term Expression of the Transduced Muscle CellsInjected into the Joints of Adult SCID Mice

5×10⁵ transduced immortalized muscle cells infected with theAV-HCMV-LacZ vector (MOI=25) were incubated with FLMs, resuspended in 10μl of HBSS and injected into the knees of 12 adult SCID(immunodeficient) mice. Three mice were sacrificed at 5, 15, 25 and 35days post-injection. The knees were first decalcified at 4° C. in a50:50 dilution of 0.5 M EDTA and 1 M glucose solution for 3 days. Theywere then flash-frozen and cryostat-sectioned in their entirety in 10 μmthick slices. β-galactosidase expression was analyzed using X-galhistochemistry and FLM localization was visualized using fluorescentmicroscopy (Nikon Optiphot-2). SCID mice were used in this experiment tobypass potential immunological complications associated with the firstgeneration adenovirus. Adult mice were used to ensure that the abilityof myoblasts to adhere to intra-articular structures was reproducible inthe mature knee.

This study was subdivided into three interrelated sections, which aredescribed in greater detail hereinbelow. First, the primary myoblasts,immortalized myoblasts, primary synovial cells and immortalized synovialcells were characterized in vitro for their efficiency of viraltransduction using a first generation adenoviral vector. This phasecompared myoblasts to cell types already being used as gene deliveryvehicles to the joint.

The early fate (5 days post-injection) of the injected muscle cells intothe joint was analyzed. This second phase included the evaluation of theviability of the injected cells in vivo, determination of the internalstructures of the joint to which the myoblasts adhered, evaluation ofmyoblast mediated gene transfer of β-galactosidase, and characterizationof the ability of the muscle cells to differentiate into myotubes andmyofibers in the joint.

The third phase analyzed the long term expression of myoblast mediatedgene transfer in the adult knee. Briefly, a myoblast cell culture wasisolated from a muscle biopsy. These cells were then transduced in vitroby an adenoviral vector carrying the LacZ reporter gene. The transducedmyoblasts were injected into the knee joint.

I) In vitro analysis: All four cell types: primary synovial cells,immortalized synovial cells, primary myoblasts and immortalizedmyoblasts were isolated, grown, and transduced with the AV-HCMV-LacZvector in vitro. Expression of β-galactosidase was observed in synovialcells (FIGS. 3A and 3B) and in myoblasts (FIGS. 3C and 3D) at 2 days(FIGS. 3A and 3C) and 6 days (FIGS. 3B and 3D) post-infection. However,when transduced immortalized myoblasts were allowed to fuse in culture,multiple fused multinucleated myotubes expressing β-galactosidase wereseen (FIG. 3D). The positive desmin fluorescent staining (greenfluorescence—FITC) validated the high myogenicity index of the myoblastculture (FIG. 3E).

When the different cell types were analyzed in the LacZ assay at twodays after infection, 2.5 to 5.0 picograms of β-galactosidase per onemillion cells were produced by the primary synovial, pure synovial, andprimary muscle cells. In contrast, the immortalized, transducedmyoblasts produced over 20 picograms of β-galactosidase per one millioncells. This was significantly higher (paired T-tests) than the othercell types, p<0.05 (FIG. 3F). In addition, the transduction of myoblastand synovial cells with an adenovirus which carry the expression ofIL-1Ra lead also to a higher production of IRAP by the myoblast cellsthan the synovial cells (FIG. 4).

II) Determination of the early fate of the injected muscle cells intothe joint: The histologic examination of newborn rabbit knee sectionsdemonstrated that the injected myoblasts had fused with most of thestructures of the knee. Transplantation of transduced synovial cellsinto the knee produced β-galactosidase, but only in some parts of thesynovial lining (S) of the joint. However, by injecting primarymyoblasts, numerous tissues of the joint, i.e., synovium (S), meniscus(M) and ligament were found to contain cells expressing β-galactosidase.With desmin staining (green fluorescence—FITC), very few myotubes weredetected in the injected joint with primary myoblasts.

The poor level of gene transfer mediated by injection of primarymyoblasts may be attributed to a decrease in the number of muscle cellsand also to the heterogeneous population of cells in these cultures(fibroblasts, adipocytes, etc.). In fact, a better gene complementationwas achieved in muscle cells in vitro by using the immortalized myoblastculture compared with the primary myoblast culture containing other celltypes. In order to validate this hypothesis, myoblasts from animmortalized cell line were then investigated using a similar ex vivoapproach.

The transplantation of immortalized myoblasts produced large patches ofmuscle cells expressing β-galactosidase in the synovial lining adjacentto the patella. With desmin immunofluorescence staining, multiple fusedmyotubes and myofibers were visualized in the same area. Some of themyotubes were seen longitudinally, and others were seen in cross section(rounded circular structures). These myotubes and myofibers weredefinitely formed by the fusion of the transplanted myoblasts due to theco-localization of FLMs and desmin positive cells. Moreover, someinjected myoblasts adhered to the patellar ligament and expressedβ-galactosidase within the striated ligamentous structures. In this samearea, multiple long myotubes stained for desmin were depicted, thusdemonstrating the presence of myogenic cells at this location.

Most of the knees showed a propensity for the myoblasts to congregateand fuse in the joint capsule of the femoral lateral recess. In contrastto the synovial cell and primary myoblast injections, immortalizedmyoblasts were able to produce large patches of myotubes and myofibersexpressing β-galactosidase in the joint capsule. Large disorganizedpatterns of myofibers expressing desmin co-localized with FLMs were alsoobserved at the same location. This was distinctly different from thenormal extra-capsular in vivo muscles that also stained positive fordesmin, but were without FLMs. On higher magnification, cross sectionsof large diameter desmin positive muscle fibers co-localized with FLMswere seen in this joint capsule (Day et al, 1997 J. Orthop. Res., 15,894-903).

In parts of the cruciate ligaments located in the femoral notch (FN),the presence of myoblasts expressing β-galactosidase was observedfollowing transplantation of immortalized myoblasts into the joint.However, smaller and more irregularly shaped desmin positive cells weredetected, suggesting the presence of myoblasts that had not yetdifferentiated into myofibers. At higher magnification, transversesections of small myotubes containing FLMs were still visualized in thefemoral notch.

III) Determination of the Long Term Expression of Muscle Cells Injectedinto the Adult Mouse Knee

When transduced immortalized myoblasts were injected into the knee ofadult SCID mice, myoblast mediated gene transfer of the LacZ reportergene was also seen in various structures in the knee including synovium,capsule, and tissues in the femoral notch. β-galactosidase productionco-localized with FLMs was seen at 35 days post-injection. In the 35 daygroup, large aggregates of rounded structures suggestive of myotubes andmyofibers producing β-galactosidase were seen in the femoral notch,synovium, and joint capsule.

The direct injection of the transduced muscle cells into theintraarticular structures leads to a high level of gene transfer in themeniscus and the anterior cruciate ligament. FIGS. 5A-5D show theresults of myoblast-mediated ex vivo gene transfer in rabbit meniscus.Myoblasts transduced with an adenovirus vector carrying the geneencoding β-galactosidase (LacZ) were injected into rabbit meniscus.FIGS. 5A and 5B show the expression of LacZ in the meniscus followinginjection and expression of β-galactosidase. FIG. 5C shows that LacZstaining is co-localized with fluorescent latex microspheres in theinjected area. FIG. 5D shows the expression of desmin, a myogenic marker(green fluorescence).

FIGS. 6A and 6B show the results of myoblast-mediated ex vivo genetransfer into rabbit ACL ligament. Myoblasts transduced with anadenovirus vector carrying the gene encoding β-galactosidase (LacZ) wereinjected into rabbit ligament. FIGS. 6A and 6B show the expression ofLacZ in the ligament following injection and expression ofβ-galactosidase. FIG. 6C shows that LacZ staining is co-localized withfluorescent latex microspheres in the injected area. FIG. 6D shows theexpression of desmin, a myogenic marker (green fluorescence), revealingthe presence of muscle cells in the ligament.

In vitro data detail the effects of numerous growth factors onfibroblast proliferation and collagen production. In fact, according tothe present invention, platelet-derived growth factor (PDGF), epidermalgrowth factor (EGF), transforming growth factor α (TGF-α), and basicfibroblast growth factor (bFGF), were observed to improve theproliferation of the meniscal fibrochondrocytes and their expression ofcollagen and non-collagen proteins.

Regardless of which growth factor is employed for meniscal healing, thecardinal issue of protein delivery must be addressed. Directintrameniscal of the recombinant growth factor protein injections areunlikely to produce sustained levels without the need for multipleinjections, a scenario that is not clinically appropriate. Efficient andsustained delivery of desired growth factors may be best accomplished bygene delivery. As disclosed herein, muscle cell-mediated ex vivo genedelivery offers the possibility of sustained, high level geneexpression.

The muscle cell-mediated ex vivo approach was further employed todeliver marker genes to the rabbit meniscus. The results demonstratedthat muscle-derived cells can be used as a gene delivery vehicle to themeniscus (FIGS. 5A-5D). The ability of muscle-derived cells to be usedas a reservoir of secreting molecules to enhance meniscal healing, andthe ability of some populations of muscle-derived cells to differentiateinto various lineages allow such cells to participate in the meniscalhealing process. These findings may lead to novel therapies for meniscalinjuries, preventing significant morbidity from these chronicallydisabling injuries.

In addition, the capacity of meniscal cartilage for healing in theavascular central portion of the meniscus is very limited, possiblydirectly related to the blood supply which exists only in the peripheralthird of the meniscus. Experimental studies have shown that the healingprocess in the central part of the meniscus might be promoted by somechemotactic or mitogenic stimuli delivered by the fibrin clot orsynovial tissue. The use of vascular endothelial growth factor (VEGF),which promotes angiogenesis, is likely to be helpful to improve meniscalhealing. FIG. 12 presents a schematic of an adeno-associated viralconstruct to carry the expression of VEGF for delivery via muscle-basedgene therapy and tissue engineering.

IV) Use of Muscle Cells to Deliver Genes in a Cartilage Defect

Articular cartilage has a limited capacity to repair after injury.Defects of articular cartilage that do not penetrate the subchondralbone can not heal efficiently and result in the degeneration ofarticular cartilage. On the other hand, injuries which penetrate thesubchondral bone result in the formation of fibrocartilage orhyalin-like cartilage that are different from the normal articularcartilage, and eventually lead to the degeneration of joint cartilage.According to this invention, muscle cells were used to deliver genes ina cartilage defect (FIG. 14).

Moreover, the transposition of a muscle flap into a cartilage defect canalso be used to improve cartilage healing (FIG. 13). It is envisionedthat muscle based gene therapy and tissue engineering according to thepresent invention can be used to improve the healing of articularcartilage defect. The use of adeno-associated virus encoding moleculessuch as BMP-2, VEGF, and IGF-1 can be used to further improve thehealing of the articular cartilage, as depicted in the schematicrepresentation of FIG. 12.

In addition, many acquired musculoskeletal conditions would be amenableto new vehicles that safely and efficiently deliver genes and theirexpression, namely, degenerative arthritis, cartilage damage, ligamentdamage, delayed unions or non-unions in fractures, osteosarcoma andvarious rheumatoid diseases. Since the knee joint sustains many of theseconditions, improved gene therapy to the joint, for example, as a drugdelivery system, is an important and needed achievement in the art, andone that is readily provided by the present invention.

Example 10

This example sets forth experiments that were performed to demonstratethe use and advantages of myoblast mediated gene transfer to amelioratea bone defect.

A myoblast cell line isolated from transgenic mdx mice carrying athermolabile SV40 T-antigen under the control of an inducible promoter(J. E. Morgan et al., 1994, Dev. Biol., 162:486-498) was used. Theimmortalized mdx cell line proliferated indefinitely at 33° C. withgamma interferon and underwent normal differentiation at 37-39° C.without gamma interferon. These myoblasts were kept in cell culture andinfected with the adenoviral-lacZ vector (MOI=25). The cells were alsoincubated with fluorescent latex microspheres (FLMs) which served asanother marker by which the fate of these cells could be followed in thebone defect. Prior to injection, some flasks containing the transducedcells were analyzed for β-galactosidase production and desmin expressionby immunofluorescence staining.

External fixators were surgically placed in the right tibias of 8 adultrabbits. A 0.7-1 cm tibial bone defect was created in the rabbit via anosteotomy between the second and third pins of the external fixator.7×10⁶ transduced myoblasts were trypsinized and injected into the musclesurrounding the bone defect during the osteotomy. The same number oftransduced myoblasts were also injected percutaneously into the bonedefect 24 hours after the osteotomy. One osteotomized rabbit did notreceive the myoblast injections and served as a sham control. Theanimals were sacrificed at 6 days post-injection, and the entire legwith the external fixator in place was analyzed macroscopically for lacZexpression. The tissue in the defect and the surrounding muscles werethen flash frozen, cryostat sectioned, assayed for β-galactosidase byhistochemistry and for desmin by immunofluorescence as described in C.S. Day et al., 1997, J. Orthop. Res., 15:894-903). The FLMs werelocalized using fluorescent microscopy.

All of the rabbit tibial defects injected with transduced myoblastsdemonstrated β-galactosidase production macroscopically in the musclessurrounding the defect and in the defect itself. In contrast, the shamoperated control defect did not demonstrate any β-galactosidaseproduction. When the tissue in the defect was cryostat-sectioned andanalyzed microscopically for lacZ expression, many rounded cellsproducing β-galactosidase were seen in the midst of the much smallerfibroblasts. Moreover, when analyzed concomitantly under fluorescentmicroscopy, numerous FLMs were co-localized with the rounded myofibersexpressing the lacZ reporter gene. LacZ expression was also seen in themuscles surrounding the defect that had received the myoblast injectionson day 7 after the injection.

The sectioned defect tissue was also analyzed for desmin expression, andmany areas in the injected bone that expressed desmin were alsoco-localized with FLMs. Desmin staining was indicative of the presenceof muscle cells that had fused into myofibers in the non-muscle area ofthe segmental bone defect. Thus, myoblasts successfully delivered themarker gene into a bone defect, the protein product was expressed, andmyofibers had formed, thus allowing for the persistence of gene productexpression in the fused myotube cells derived from the geneticallyengineered myoblast cells injected into the bone.

Example 11

To confirm that a population of cells in skeletal muscle was capable ofdifferentiating into bone, variable populations of cells derived frommuscle were stimulated by BMP-2 protein and analyzed for osteogenicdifferentiation in vitro as described below.

Primary cell cultures were obtained from an adult mdx mouse (T. A. Randoand H. M. Blau, 1994, J. Cell. Biol., 125:1275-1287). Briefly, the mousewas euthanized by cervical dislocation and approximately 500 mg ofhindlimb muscle was immediately dissected and minced. The muscle wasenzymatically digested by serial incubation in 0.2% collagenase, dispase(2.5 units/ml) and 0.1% trypsin, each for 1 hour at 37° C. Any remainingcellular clumping was disassociated by passage through a 20 gaugeneedle. The cells were then plated onto collagen-coated flasks in F10Ham (Gibco BRL, Gaithersburg, Md.), supplemented to contain 10% horseserum, 10% fetal bovine serum, 1% penicillin/streptomycin, and bFGF(human recombinant, Life Technologies). Cells were subdivided accordingto their cellular adhesion characteristics by serial passage of cellsupernatant to a new flask after approximately 15-20% of the cellsadhered to the flask. This technique, termed preplating is described inExample 1, Purification of Primary Myoblasts, herein. This procedure wasrepeated six times, yielding six preplates. Cells adhering to the flasksearlier remained in the lower preplates, while those passed along in theserial supernatants adhered in the higher preplates. Fibroblasts adheremore rapidly than do myoblasts under the conditions of this procedure;hence, the higher preplates are enriched for myoblasts.

The different subpopulations of cells were plated at 2×10⁴/well in 12well plates. Cells from each preplate were incubated in medium or inmedium supplemented to contain 50 ng/ml or 200 ng/ml BMP. BMP was addedto the appropriate wells at 1, 3 and 5 days after plating. A cell lysatewas obtained 24 hours after the final BMP-2 stimulation for the assay ofalkaline phosphatase activity. The cells found to be the most responsivewere re-examined under stimulation with 100 ng/ml of BMP-2 stimulationover the same time course. To study the effect of BMP-2 stimulation overtime, a portion of the cells was removed after each addition of BMP-2for the analysis of desmin expression and ALP activity.

The analysis of desmin expression in the cells was performed by standardimmunohistochemical techniques. Cells were fixed with cold methanol for1 minute, rinsed in phosphate buffered saline (PBS) and blocked with 10%horse serum for 1 hour. The cells were then incubated with a 1/100dilution of mouse anti-desmin antibody (Sigma) for 6-10 hours at 37° C.Following the rinses with PBS, the cells were incubated with a 1/100dilution of anti-mouse antibody conjugated to Cy3 fluorescent marker(Sigma) for 1 hour. Staining was then assessed with an immunofluorescentmicroscope (Nikon). Data were quantified by examining five differentfields at 10× magnification and counting the positive and negative cellsto establish a ratio. Alkaline phosphatase activity (in U/L) wasdetermined by the hydrolysis of p-nitrophenyl phosphate to p-nitrophenoland inorganic phosphate using a commercially available reagent andprotocol (Sigma). When appropriate, statistical analysis was performedby ANOVA testing for statistical difference at greater than 95%confidence intervals.

The results showed that prior to stimulation with BMP-2, the differentsubpopulations of muscle-derived cells were characterized by desminstaining. For the cell platings, cells in preplate #1 stained 3% desminpositive versus 70.5% positive cells in preplate #6. The percentage ofdesmin positive cells increased with statistically significantincrements between preplate #3 and preplate #5, and between preplate #5and preplate #6. Of interest, the cells having the highest level ofdesmin positivity tended to appear round and divide more slowly thanthose in earlier preplates.

After stimulation with BMP-2, the subpopulations of cells were testedfor the induction of alkaline phosphatase, a biochemical indicator ofosteoblastic activity (A. I. Caplan, 1991, J.Orthop. Res., 9:641-650). Amouse-derived population of stromal cells was used as a positivecontrol. Muscle-derived cells not receiving BMP-2 did not express ALPactivity. Only the subpopulation obtained from preplate #6 showed anincrease in ALP activity in response to stimulation with BMP-2. Adose-dependent trend was seen when comparing data for preplate #6 usingBMP-2 at a concentration of 50 ng/ml versus 200 ng/ml (FIG. 9).

Corresponding to the increase in ALP activity in response to BMP-2stimulation in preplate #6, the percentage of desmin-staining cellsdecreased (FIG. 10). After a single dose of BMP-2, the percentage ofdesmin-positive cells in preplate #6 decreased from 70.5% to 47%.Additional doses of 100 ng/ml of BMP-2 did not cause as much of adecrease; the level of desmin positive cells remained at approximately40%.

In addition, different populations of muscle-derived cells were isolatedfrom a human muscle biopsy and were purified by the preplate technique.When stimulated with BMP-2 as described above, cells from some of thepreplates (i.e., pp2, pp3, and pp4 to a lesser extent) expressedalkaline phosphatase (marker for pre-osteoblasts). Therefore, accordingto the invention, human muscle-derived cells can be isolated andpurified and shown to have stem cell characteristics.

Cells isolated from skeletal muscle are capable of responding torecombinant human (rhBMP-2) both in vitro and in vivo. Primary rodentmyogenic cells in cell culture respond in a dose dependent fashion torhBMP-2 by producing alkaline phosphatase, an osteogenic protein.Furthermore, the purer the population of myogenic cells, as evidenced bydesmin staining, the greater the alkaline phosphatase production.Recombinant human BMP-2 inhibits myogenic differentiation as itstimulates osteoblastic differentiation of the muscle-derived cells (A.Yamaguchi et al., 1991, J. Cell Biol., 113:681-687; T. Katagiri et al.,1994, J. Cell Biol., 127:1755-1766; K. Kawasaki et al., 1998, Bone,23:223-231). Accordingly, in vitro data suggest that myogenic cells arecapable of responding to rhBMP-2 and entering an osteogenic lineage.

Primary rodent muscle-derived cells were engineered to produceintramuscular bone in vivo. The ex vivo approach was utilized totransduce the primary muscle-derived cells with an adenovirus carryingthe BMP-2 cDNA. Intramuscular injection of as little as 300,000transduced cells produced bone in severe combined immune deficient(SCID) mice. The bone produced contained osteoid and bone marrowelements as evidenced by regular histology and von Kossa formineralization. Not only did the transduced muscle cells produce BMP-2,but the injected cells also respond to BMP-2 by producing bone

Moreover, engineered muscle-derived cells within diffusable chambers(see Example 1) that preclude the entry of host cells due to the poresize, produced bone when implanted sub-cutaneously in immunodeficientmice. These results suggest that these specific populations ofmuscle-derived cells are capable of producing bone (FIG. 11). Theability of the cells to differentiate into other lineages such as bonesubstantiate the pluripotent nature of these muscle-derived cells, i.e.,muscle-derived stem cells.

As described hereinabove, the preplate technique provided a means forattaining different populations of muscle-derived cells in differentpreplates. With selective preplates of cells, bone formation wasachieved (e.g., FIG. 11), thus, strongly suggesting the presence ofmuscle stem cells. Table 6 summarizes the expression of differentmarkers by purified mouse muscle-derived cells.

TABLE 6 Cell Type Mdx pp#6 Normal pp#6 Fibroblast Desmin + + − Bcl2 + +− CD34 + + − MyoD +/− +/− − Myogenin +/− +/− − M-Cadherin −/+ −/+ −MyHCs (+) (+) −

In Table 6, Mdx pp#6 and normal pp#6 were derived from hindlimb muscleof newborn mdx and normal mice, respectively, by the preplate techniqueas described herein. “+” indicates that more than 95% of the cells inthe culture expressed high levels of antigen. “+/−” indicates thatapproximately 60% or 30% of the cells in the cultures expressed MyoD orMyogenin antigen, respectively. “−/+” indicates that less than 10% ofthe cells in the culture expressed M-Cadherin, and “(+)” indicates morethan 95% of the cells in the three-day-fusion medium expressed myosinheavy chain isoform (MyHCs).

In addition to the ex vivo approach, an adenovirus mediated direct genetransfer of BMP-2 produced large amounts of intramuscular bone.Consequently, both the in vitro and in vivo data support the hypothesisthat muscle cells may be engineered to become osteogenic cells. Theramifications of myogenic cells' capabilities to form bone are immense.In fact, the ability of the adenovirus carrying the expression of BMP-2to induce radiographic and histologic ectopic bone formation at 2, 3 and4 weeks post-injection suggests that muscle flap can be eventually usedas a biological scaffold with the ability to improve healing of hardtissue including bone and cartilage. In fact, the use of a muscle flaptreated with Adenovirus-BMP-2 may be capable of improving bone healing.

Since the use adenovirus vectors may be hindered by immune responsesagainst the vectors, the use of a less immunogenic vector has beendetermined to be required to improve bone formation by using the muscleas a biological scaffold. Therefore, adeno-associated virus has beenused as a new viral vector to improve the efficiency of gene transfer tomature skeletal muscle. This vector can be employed to deliver BMP-2into muscle flap to improve bone healing. A schematic representation ofthe plasmid use to construct an adeno-associated virus to carry theexpression of BMP-2 is presented in FIG. 12.

Muscle-based tissue engineering to produce bone may be applicable tomultiple skeletal abnormalities. One such scenario is large bone defectsresulting from trauma or oncologic resections. Muscle-derived cellscapable of bone formation may be exploited to reconstruct the bonedefect and minimize the use of autograft, allograft, and bonedistraction. A muscle flap may be able to be engineered to produce boneand, thereby, reconstruct an experimental bone defect. Both ex vivo andin vivo gene therapy techniques are amenable for bone formation and toreconstruct bone defects.

Another approach is to transform muscle, restricted to the confines of asilicone mold, into bone of desired geometry such as a proximal femur ormidshaft tibia (R. K. Khouri et al., 1991, JAMA, 266:1953). Themuscle-based approach to bone defect reconstructions is especiallyappealing in light of the often poor vascularity of traumatic andoncologic bone defects. The combination of vascularized muscle and denovo bone formation offers revolutionary possibilities for bone repair.

Example 12

This example provides a typical and highly practical application ofmuscle-derived cell mediated gene therapy and tissue engineering asprovided by the present invention for the treatment of urinary tractdysfunction in human patients. The present invention has provided theability to perform gene delivery to the lower urinary tract. Inaddition, the present invention has demonstrated that myoblast mediatedgene therapy was more successful in delivering iNOS than using directvirus or plasmid infection/transfection methods.

The direct clinical utility of the present invention offers thoseskilled in the art the ability to treat patients simply, safely andefficiently on an out-patient basis, and even in the doctor's office.For example, in a urology office, patients with stress urinaryincontinence undergo a simple needle aspiration of their muscle, forexample, the triceps, that takes less than 5 minutes.

The muscle cells are grown under the appropriate cell culture conditionsin a laboratory, preferably, a laboratory of a biotechnology center.This step takes about 1-4 weeks. The cultured muscle cells, now vastlyincreased in number, are shipped back to the treating doctor and arethen injected back into the patient in a brief, 10 minute outpatientendoscopic procedure. The injection is performed using a smallcystoscope and a cystoscopic needle. Under direct surgeon's vision, theneedle tip is inserted into the urethral sphincter mechanism and themyoblast suspension is injected into the urethral wall to cause urethralcoaptation and closure.

Cultured myoblasts can be frozen and stored indefinitely for possiblefuture use. As described, muscle-derived cells can also be used for cellmediated gene therapy with various trophic factors to augment and/orenhance the treatment and repair process in a given tissue. Similarprocedures of myoblast injection and gene therapy can also be done inthe bladder of patients with impaired bladder contractility.

The number of cells removed from the patient does not need to be large,if the cells are subsequently placed in culture where they willproliferate and increase in number prior to injection. For injection,the number of cells used can be determined by the practitioner usingroutine skill, depending on the specific injury, disease or dysfunctionbeing treated, the tissue or organ to be injected and the gene constructused. In general a lower cell number is used for gene delivery/genetherapy procedures, while a higher cell number is used for tissueengineering and bulking, i.e., on the order of about 1×10⁴ to about1×10¹⁴, preferably about 1×10⁴ to about 1×10⁶ for gene delivery. If thecell number is found to be too low to produce an effective amount ofgene product at and near the site of injection, repeat injections areeasily performed and may be administered as needed.

In the inventors' practice of the protocol of the present invention, theinjected muscle-derived cells have been found to remain at and near thesite of injection in bladder, urethra, penis, leg muscle, knee jointsand bone, for example. Further, histology of various tissuespost-injection has not shown significant scar tissue formation, even ifallogeneic cells were used. With autologous muscle-derived cells, scartissue formation was virtually nil.

At and near the injection site, the injected myoblasts fused and formedmyotubes based on the amount of surrounding space. Once the cells hadfilled up the area at the site of injection, myofibers formed and thecells no longer proliferated or grew. The multinucleated myofiberremained virtually the same size after it was formed and did notproliferate or die. Thus, on a longterm basis, the myofiber stablyproduced and secreted the gene product of the delivered, expressedencoding gene in the area of injection.

The contents of all patents, patent applications, published articles,books, reference manuals, texts and abstracts cited herein are herebyincorporated by reference in their entirety to more fully describe thestate of the art to which the present invention pertains.

The present invention has been described in detail including thepreferred embodiments thereof. However, it will be appreciated by thoseskilled in the art, upon consideration of this disclosure, thatmodifications and improvements may be made thereon without departingfrom the spirit and scope of the invention as set forth in thedescription and claims.

1. A method of augmenting skeletal muscle tissue in a subject in need thereof comprising: introducing an isolated population of cells enriched in viable, non-fibroblast, desmin-expressing, autologous, skeletal muscle-derived myoblasts into a site of injured or damaged skeletal muscle tissue of a recipient in an amount effective to augment skeletal muscle tissue, wherein the injury or damage is the result of laceration, contusion, or strain.
 2. The method according to claim 1, wherein the skeletal muscle-derived myoblasts are histocompatibly-matched with the recipient.
 3. The method according to claim 1, wherein the skeletal muscle-derived myoblasts are introduced in a composition comprising a physiologically acceptable medium.
 4. The method according to claim 1, wherein the skeletal muscle-derived myoblasts are introduced in an amount of about 10⁵ to 10⁶ cells per cm³ of tissue to be treated in a physiologically acceptable medium.
 5. The method according to claim 1, wherein a cloned population of the skeletal muscle-derived myoblasts is introduced into the recipient.
 6. The method according to claim 1, wherein the skeletal muscle-derived myoblasts are contacted with a cytokine or growth factor selected from one or more of basic fibroblast growth factor (b-FGF), insulin-like growth factor (IGF), or nerve growth factor (NGF), prior to introducing the skeletal muscle-derived myoblasts into the recipient.
 7. The method according to claim 1, wherein the skeletal muscle-derived myoblasts carry a vector containing a heterologous gene.
 8. The method according to claim 1, wherein the skeletal muscle-derived myoblasts have been obtained according to a culture method comprising: (i) plating a suspension of cells from skeletal muscle tissue in a first container, to which fibroblast cells in the muscle tissue suspension adhere; (ii) re-plating non-adherent cells from (i) in a second container after approximately 15% to 20% of the cells from the cell suspension have adhered to the first container; (iii) repeating step (ii) at least one time to enrich for an end population of viable, non-fibroblast, desmin-expressing, skeletal muscle-derived myoblasts; and (iv) isolating the enriched end population of skeletal muscle-derived myoblasts.
 9. The method according to claim 1, wherein the population of skeletal muscle-derived myoblasts is enriched to comprise 14% to 95% desmin positive cells.
 10. The method according to claim 1, wherein the population of skeletal muscle-derived myoblasts is enriched to comprise 14% to 80% desmin positive cells.
 11. The method according to claim 1, wherein the skeletal muscle-derived myoblasts are genetically unmodified.
 12. The method of claim 1, wherein the muscle injury is the result of laceration.
 13. The method of claim 1, wherein the muscle injury is the result of contusion.
 14. The method of claim 1, wherein the muscle injury is the result of strain. 